Recombinant polymerases with increased phototolerance

ABSTRACT

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional utility patent applicationclaiming priority to and benefit of the following prior provisionalpatent application: U.S. Ser. No. 61/593,569, filed Feb. 1, 2012,entitled “RECOMBINANT POLYMERASES WITH INCREASED PHOTOTOLERANCE” bySatwik Kamtekar et al., which is incorporated herein by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

The invention relates to modified DNA polymerases for single moleculesequencing. The polymerases include modified recombinant polymerasesthat display a reduced susceptibility to photodamage. The invention alsorelates to methods for amplifying nucleic acids and to methods fordetermining the sequence of nucleic acid molecules using suchpolymerases.

BACKGROUND OF THE INVENTION

DNA polymerases replicate the genomes of living organisms. In additionto this central role in biology, DNA polymerases are also ubiquitoustools of biotechnology. They are widely used, e.g., for reversetranscription, amplification, labeling, and sequencing, all centraltechnologies for a variety of applications such as nucleic acidsequencing, nucleic acid amplification, cloning, protein engineering,diagnostics, molecular medicine, and many other technologies.

Because of the importance of DNA polymerases, they have been extensivelystudied. This study has focused, e.g., on phylogenetic relationshipsamong polymerases, structure of polymerases, structure-function featuresof polymerases, and the role of polymerases in DNA replication and otherbasic biological processes, as well as ways of using DNA polymerases inbiotechnology. For a review of polymerases, see, e.g., Hübscher et al.(2002) “Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol.71: 133-163, Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1): reviews 3002.1-3002.4, Steitz (1999)“DNA polymerases: structural diversity and common mechanisms” J BiolChem 274:17395-17398, and Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol. Chem. 276(47):43487-90. Crystal structures have been solved for many polymerases,which often share a similar architecture. The basic mechanisms of actionfor many polymerases have been determined.

A fundamental application of DNA technology involves various labelingstrategies for labeling a DNA that is produced by a DNA polymerase. Thisis useful in DNA sequencing, microarray technology, SNP detection,cloning, PCR analysis, and many other applications. Labeling is oftenperformed in various post-synthesis hybridization or chemical labelingschemes, but DNA polymerases have also been used to directly incorporatevarious labeled nucleotides in a variety of applications, e.g., via nicktranslation, reverse transcription, random priming, amplification, thepolymerase chain reaction, etc. See, e.g., Giller et al. (2003)“Incorporation of reporter molecule-labeled nucleotides by DNApolymerases. I. Chemical synthesis of various reporter group-labeled2′-deoxyribonucleoside-5′-triphosphates” Nucleic Acids Res.31(10):2630-2635, Augustin et al. (2001) “Progress towardssingle-molecule sequencing: enzymatic synthesis ofnucleotide-specifically labeled DNA” J. Biotechnol. 86:289-301, Tonon etal. (2000) “Spectral karyotyping combined with locus-specific FISHsimultaneously defines genes and chromosomes involved in chromosomaltranslocations” Genes Chromosom. Cancer 27:418-423, Zhu and Waggoner(1997) “Molecular mechanism controlling the incorporation of fluorescentnucleotides into DNA by PCR” Cytometry, 28:206-211, Yu et al. (1994)“Cyanine dye dUTP analogs for enzymatic labeling of DNA probes” NucleicAcids Res. 22:3226-3232, Zhu et al. (1994) “Directly labeled DNA probesusing fluorescent nucleotides with different length linkers” NucleicAcids Res. 22:3418-3422, and Reid et al. (1992) “Simultaneousvisualization of seven different DNA probes by in situ hybridizationusing combinatorial fluorescence and digital imaging microscopy” Proc.Natl. Acad. Sci. USA, 89:1388-1392.

DNA polymerase mutants have been identified that have a variety ofuseful properties, including altered nucleotide analog incorporationabilities relative to wild-type counterpart enzymes. For example,Vent^(A488L) DNA polymerase can incorporate certain non-standardnucleotides with a higher efficiency than native Vent DNA polymerase.See Gardner et al. (2004) “Comparative Kinetics of Nucleotide AnalogIncorporation by Vent DNA Polymerase” J. Biol. Chem. 279(12):11834-11842and Gardner and Jack “Determinants of nucleotide sugar recognition in anarchaeon DNA polymerase” Nucleic Acids Research 27(12):2545-2553. Thealtered residue in this mutant, A488, is predicted to be facing awayfrom the nucleotide binding site of the enzyme. The pattern of relaxedspecificity at this position roughly correlates with the size of thesubstituted amino acid side chain and affects incorporation by theenzyme of a variety of modified nucleotide sugars.

Additional modified polymerases, e.g., modified polymerases that displayimproved properties useful for single molecule sequencing (SMS) andother polymerase applications (e.g., DNA amplification, sequencing,labeling, detection, cloning, etc.), are desirable. The presentinvention provides new recombinant DNA polymerases with desirableproperties, including increased resistance to photodamage. Otherexemplary properties include exonuclease deficiency, altered cofactorselectivity, increased yield, increased thermostability, increasedaccuracy, increased speed, increased readlength, and the like. Alsoincluded are methods of making and using such polymerases, as well asmany other features that will become apparent upon a complete review ofthe following.

SUMMARY OF THE INVENTION

Modified DNA polymerases can find use in such applications as, e.g.,single-molecule sequencing (SMS), genotyping analyses such as SNPgenotyping using single-base extension methods, sample preparation, andreal-time monitoring of amplification, e.g., RT-PCR. Among otheraspects, the invention provides compositions comprising recombinantpolymerases that comprise mutations which confer properties which can beparticularly desirable for these applications. These properties can,e.g., increase enzyme (and therefore assay) robustness, facilitatereadout accuracy, or otherwise improve polymerase performance. Alsoprovided by the invention are methods of generating such modifiedpolymerases and methods in which such polymerases can be used to, e.g.,sequence a DNA template and/or make a DNA.

One general class of embodiments provides a composition comprising arecombinant Φ29-type DNA polymerase, which recombinant polymerasecomprises one or more mutation selected from the group consisting of anamino acid substitution at position 131, an amino acid substitution atposition 132, a K135Q substitution, a K135S substitution, an H149Dsubstitution, a Q183F substitution, a G197D substitution, a G197Esubstitution, an I201E substitution, a K206E substitution, an A437Nsubstitution, and a D510S substitution, wherein identification ofpositions is relative to wild-type Φ29 polymerase (SEQ ID NO:1).Exemplary mutations at positions 131 and 132 include, e.g., K131E,K131Q, and K132Q. Optionally, the recombinant polymerase is moreresistant to photodamage than is a wild-type polymerase or a parentalpolymerase lacking the one or more mutations.

The polymerase can also include mutations at additional positions. Forexample, the polymerase can include one or more mutation or combinationof mutations selected from the group consisting of an amino acidsubstitution at position 253, an amino acid substitution at position375, an amino acid substitution at position 484, an amino acidsubstitution at position 512, an amino acid substitution at position510, an amino acid substitution at position 148, an amino acidsubstitution at position 224, an amino acid substitution at position239, an amino acid substitution at position 250, an amino acidsubstitution at position 437, an amino acid substitution at position235, an amino acid substitution at position 515, an amino acidsubstitution at position 141, an amino acid substitution at position142, an amino acid substitution at position 504, an amino acidsubstitution at position 508, an amino acid substitution at position513, an amino acid substitution at position 523, an amino acidsubstitution at position 536, an amino acid substitution at position539, an amino acid substitution at position 205, an amino acidsubstitution at position 472, an amino acid substitution at position 437and an amino acid substitution at position 253, and an amino acidsubstitution at position 508 and an amino acid substitution at position510, wherein identification of positions is relative to SEQ ID NO:1.

The polymerase optionally includes one or more mutation or combinationof mutations selected from the group consisting of A437G and L253H,A437G and L253C, V250A and L253H, A437G, D235E, E515Q, E515P, E515K,V250A, V250I, Y148I, Y224K, E239G, V141K, L142K, E508K, E508K and D510S,K536Q, K539Q, K205E, K205D, K205A, K472A, E375Y, K512Y, A484E, L253A,L253C, L253S, L253H, and D510K, wherein identification of positions isrelative to SEQ ID NO:1. In one class of embodiments, the recombinantpolymerase comprises E375Y, A484E, and K512Y substitutions, whereinidentification of positions is relative to SEQ ID NO:1.

Optionally, the polymerase comprises mutations at two or more, three ormore, four or more, five or more, or even six or more of the indicatedpositions. Exemplary combinations of mutations include K131E, Y148I,Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y, andE515Q; K135Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,D510K, K512Y, and E515Q; K131E, Y148I, Y224K, D235E, E239G, V250A,L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y148I, Y224K,E239G, L253S, E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y148I,Q183F, D235E, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, andE515Q; Y148I, Y224K, E239G, V250I, L253H, E375Y, A437G, A484E, D510K,and K512Y; Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,D510K, K512Y, and E515Q; K131E, Y148I, Y224K, D235E, E239G, L253H,E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y148I, Y224K, D235E,E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y148I,Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, and K512Y;Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, andK512Y; K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A484E, D510K,and K512Y; K135Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A484E,D510K, and K512Y; Y148I, Y224K, E239G, L253H, E375Y, A437G, A484E,D510K, and K512Y; K131E, K135Q, V141K, L142K, Y148I, Y224K, E239G,V250I, L253A, E375Y, A437G, A484E, E508K, D510K, K512Y, E515Q, andK536Q; K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,E508K, D510K, K512Y, and E515Q; and K131Q, Y148I, Y224K, E239G, V250I,L253A, E375Y, A484E, D510K, and K512Y; wherein identification ofpositions is relative to SEQ ID NO:1.

Additional exemplary mutations and combinations are described herein orcan be formed from those disclosed herein, and polymerases includingsuch combinations are also features of the invention.

The recombinant polymerase can be a modified recombinant Φ29 polymerase.Thus, in one class of embodiments, the recombinant polymerase is atleast 70% identical to wild-type Φ29 polymerase (SEQ ID NO:1), forexample, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or even at least 99% identical to wild-type Φ29 polymerase(SEQ ID NO:1). As another example, the recombinant polymerase can be amodified recombinant M2Y polymerase. Thus, in one class of embodiments,the recombinant polymerase is at least 70% identical to wild-type M2Ypolymerase (SEQ ID NO:2), for example, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or even at least 99% identical towild-type M2Y polymerase (SEQ ID NO:2). In other exemplary embodiments,the recombinant polymerase is a recombinant B103, GA-1, PZA, Φ15, BS32,Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17polymerase.

The recombinant polymerase optionally comprises one or more exogenousfeatures, e.g., at the C-terminal and/or N-terminal region of thepolymerase, for example, a polyhistidine tag (e.g., a His 10 tag) or abiotin ligase recognition sequence. As a few examples, the polymerasecan include a C-terminal polyhistidine tag, a C-terminal polyhistidinetag and biotin ligase recognition sequence, or an N-terminalpolyhistidine tag and biotin ligase recognition sequence and aC-terminal polyhistidine tag.

A related general class of embodiments provides a composition comprisinga recombinant DNA polymerase, which recombinant polymerase comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:27-46 or a conservative or substantially identical variant thereof.

A composition comprising a recombinant polymerase of the invention canalso include a nucleotide analog, e.g., a phosphate-labeled nucleotideanalog. The analog optionally comprises a fluorophore. The analog cancomprise three phosphate groups, or it can comprise four or morephosphate groups, e.g., 4-7 phosphate groups (that is, the analog can bea tetraphosphate, pentaphosphate, hexaphosphate, or heptaphosphateanalog). In one class of embodiments, the composition includes anucleotide analog (e.g., a phosphate-labeled nucleotide analog) and aDNA template, and the polymerase incorporates the nucleotide analog intoa copy nucleic acid in response to the DNA template. The composition canbe present in a DNA sequencing system, e.g., a zero-mode waveguide(ZMW). The recombinant polymerase can be immobilized on a surface, forexample, on a surface of a zero-mode waveguide, preferably in an activeform.

In one aspect, the invention provides methods of sequencing a DNAtemplate. In the methods, a reaction mixture that includes the DNAtemplate, a replication initiating moiety that complexes with or isintegral to the template, one or more nucleotides and/or nucleotideanalogs, and a recombinant polymerase of the invention (e.g., arecombinant Φ29-type DNA polymerase) is provided. The polymerase iscapable of replicating at least a portion of the template using themoiety in a template-dependent polymerization reaction. The reactionmixture is subjected to a polymerization reaction in which therecombinant polymerase replicates at least a portion of the template ina template-dependent manner, whereby the one or more nucleotides and/ornucleotide analogs are incorporated into the resulting DNA. A timesequence of incorporation of the one or more nucleotides and/ornucleotide analogs into the resulting DNA is identified.

The nucleotide analogs used in the methods can comprise a first analogand a second analog (and optionally third, fourth, etc. analogs), eachof which comprise different fluorescent labels. The differentfluorescent labels can optionally be distinguished from one anotherduring the step in which a time sequence of incorporation is identified.Optionally, subjecting the reaction mixture to a polymerization reactionand identifying a time sequence of incorporation are performed in a zeromode waveguide. Essentially all of the features noted for thecompositions herein apply to these methods as well, as relevant.

In a related aspect, the invention provides methods of making a DNA. Inthe methods, a reaction mixture is provided that includes a template, areplication initiating moiety that complexes with or is integral to thetemplate, one or more nucleotides and/or nucleotide analogs, and arecombinant polymerase of the invention (e.g., a recombinant Φ29-typeDNA polymerase). The polymerase is capable of replicating at least aportion of the template using the moiety in a template-dependentpolymerase reaction. The mixture is reacted such that the polymerasereplicates at least a portion of the template in a template-dependentmanner, whereby the one or more nucleotides and/or nucleotide analogsare incorporated into the resulting DNA. The reaction mixture isoptionally reacted in a zero mode waveguide. The methods optionallyinclude detecting incorporation of at least one of the nucleotidesand/or nucleotide analogs. Essentially all of the features noted for thecompositions herein apply to these methods as well, as relevant.

In one aspect, the invention provides methods of making a recombinantpolymerase. In the methods, a parental polymerase (e.g., a wild-type orother Φ29-type polymerase) is mutated at one or more of the positionsdescribed herein (e.g., one or more of positions K131, K132, K135, V141,L142, Y148, H149, Q183, G197, 1201, K205, K206, Y224, D235, E239, V250,L253, E375, A437, K472, A484, 1504, E508, D510, K512, L513, E515, D523,K536, and K539, where identification of positions is relative to SEQ IDNO:1). Optionally, one or more property of the recombinant polymerase(e.g., resistance to photodamage) is assessed and compared to that forthe parental polymerase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an alignment between the amino acid sequences ofwild-type M2Y polymerase (SEQ ID NO:2) and wild-type Φ29 polymerase (SEQID NO:1).

FIG. 2 depicts the structure of A488dA4P.

FIG. 3 Panels A and B schematically illustrate an exemplary singlemolecule sequencing by incorporation process in which the compositionsof the invention provide particular advantages.

FIG. 4 presents a fluorescence time trace for a ZMW, showing pulsesrepresenting incorporation of different nucleotide analogs. The insetschematically illustrates the catalytic cycle for polymerase-mediatedextension; the box indicates the portion of the catalytic cycle thatcorresponds to the pulse when sequencing is performed withphosphate-labeled nucleotide analogs.

FIG. 5 depicts the electrostatic surface of Φ29 polymerase with a boundphosphate-labeled hexaphosphate analog.

FIG. 6 depicts the location of exemplary residues that can be mutated toreduce positive surface charge of Φ29 polymerase without loss ofsequence performance.

FIG. 7 provides exemplary polymerase mutations and combinations thereofin accordance with the invention. Positions of the mutations areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1)where the name of the polymerase includes “Phi29” or relative to awild-type M2Y polymerase (SEQ ID NO:2) where the name of the polymeraseincludes “M2.”

FIG. 8 shows a view in the vicinity of residues 253 and 437 of arecombinant Φ29 polymerase including D12A, D66A, Y224K, E239G, L253H,E375Y, A437G, A484E, D510K, and K512Y substitutions.

Schematic figures are not necessarily to scale.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a protein”includes a plurality of proteins; reference to “a cell” includesmixtures of cells, and the like.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “nucleic acid” or “polynucleotide” encompasses any physicalstring of monomer units that can be corresponded to a string ofnucleotides, including a polymer of nucleotides (e.g., a typical DNA orRNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotidescomprising nucleotides that are not typical to biological RNA or DNA,such as 2′-O-methylated oligonucleotides), and the like. A nucleic acidcan be e.g., single-stranded or double-stranded. Unless otherwiseindicated, a particular nucleic acid sequence of this inventionencompasses complementary sequences, in addition to the sequenceexplicitly indicated.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can additionally comprisenon-amino acid elements such as labels, quenchers, blocking groups, orthe like and can optionally comprise modifications such asglycosylation, biotinylation, or the like. The amino acid residues ofthe polypeptide can be natural or non-natural and can be unsubstituted,unmodified, substituted or modified.

An “amino acid sequence” is a polymer of amino acid residues (a protein,polypeptide, etc.) or a character string representing an amino acidpolymer, depending on context.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

Numbering of a given amino acid or nucleotide polymer “corresponds tonumbering of” or is “relative to” a selected amino acid polymer ornucleic acid when the position of any given polymer component (aminoacid residue, incorporated nucleotide, etc.) is designated by referenceto the same residue position in the selected amino acid or nucleotidepolymer, rather than by the actual position of the component in thegiven polymer. Similarly, identification of a given position within agiven amino acid or nucleotide polymer is “relative to” a selected aminoacid or nucleotide polymer when the position of any given polymercomponent (amino acid residue, incorporated nucleotide, etc.) isdesignated by reference to the residue name and position in the selectedamino acid or nucleotide polymer, rather than by the actual name andposition of the component in the given polymer. Correspondence ofpositions is typically determined by aligning the relevant amino acid orpolynucleotide sequences. For example, residue K221 of wild-type M2Ypolymerase (SEQ ID NO:2) is identified as position Y224 relative towild-type Φ29 polymerase (SEQ ID NO:1); see, e.g., the alignment shownin FIG. 1. Similarly, residue L138 of wild-type M2Y polymerase (SEQ IDNO:2) is identified as position V141 relative to wild-type Φ29polymerase (SEQ ID NO:1), and an L138K substitution in the M2Ypolymerase is thus identified as a V141K substitution relative to SEQ IDNO:1. Amino acid positions herein are generally identified relative toSEQ ID NO:1 unless explicitly indicated otherwise.

The term “recombinant” indicates that the material (e.g., a nucleic acidor a protein) has been artificially or synthetically (non-naturally)altered by human intervention. The alteration can be performed on thematerial within, or removed from, its natural environment or state. Forexample, a “recombinant nucleic acid” is one that is made by recombiningnucleic acids, e.g., during cloning, DNA shuffling or other procedures,or by chemical or other mutagenesis; a “recombinant polypeptide” or“recombinant protein” is, e.g., a polypeptide or protein which isproduced by expression of a recombinant nucleic acid.

A “Φ29-type DNA polymerase” (or “phi29-type DNA polymerase”) is a DNApolymerase from the Φ29 phage or from one of the related phages that,like Φ29, contain a terminal protein used in the initiation of DNAreplication. Φ29-type DNA polymerases are homologous to the Φ29 DNApolymerase (e.g., as listed in SEQ ID NO:1); examples include the B103,GA-1, PZA, (1)15, BS32, M2Y (also known as M2), Nf, G1, Cp-1, PRD1, PZE,SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, and AV-1 DNA polymerases, as wellas chimeras thereof. A modified recombinant Φ29-type DNA polymeraseincludes one or more mutations relative to naturally-occurring wild-typeΦ29-type DNA polymerases, for example, one or more mutations thatincrease phototolerance, alter interaction with and/or incorporation ofnucleotide analogs, and/or alter another polymerase property, and mayinclude additional alterations or modifications over the wild-typeΦ29-type DNA polymerase, such as one or more deletions, insertions,and/or fusions of additional peptide or protein sequences (e.g., forimmobilizing the polymerase on a surface or otherwise tagging thepolymerase enzyme).

A variety of additional terms are defined or otherwise characterizedherein.

DETAILED DESCRIPTION

One aspect of the invention is generally directed to compositionscomprising a recombinant polymerase, e.g., a recombinant Φ29-type DNApolymerase, that includes one or more mutations (e.g., amino acidsubstitutions, deletions, or insertions) as compared to a referencepolymerase, e.g., a wild-type Φ29-type polymerase. Depending on theparticular mutation or combination of mutations, the polymerase exhibitsone or more properties that find use in, e.g., single moleculesequencing applications or nucleic acid amplification. Exemplaryproperties exhibited by various polymerases of the invention includeincreased resistance to photodamage, a reduction in the rate of one ormore steps of the polymerase kinetic cycle (resulting from, e.g.,enhanced interaction of the polymerase with nucleotide analog, enhancedmetal coordination, etc.), increased closed complex stability, analtered branching fraction, reduced or eliminated exonuclease activity,altered cofactor selectivity, and increased processivity, yield,thermostability, accuracy, speed, and/or readlength, as well as otherfeatures that will become apparent upon a complete review of the presentdisclosure. The polymerases can include one or more exogenous orheterologous features, e.g., at the N- and/or C-terminal regions of thepolymerase. Such features find use not only for purification of therecombinant polymerase and/or immobilization of the polymerase to asubstrate, but can also alter one or more properties of the polymerase.

Among other aspects, the present invention provides new polymerases thatincorporate nucleotide analogs, such as dye labeled phosphate labeledanalogs, into a growing template copy during DNA amplification. Thesepolymerases are modified such that they have one or more desirableproperties, for example, increased resistance to photodamage, decreasedbranching fraction formation when incorporating the relevant analogs,improved DNA-polymerase stability or processivity, reduced exonucleaseactivity, increased thermostability and/or yield, altered cofactorselectivity, improved accuracy, speed, and/or readlength, and/or alteredkinetic properties as compared to corresponding wild-type or otherparental polymerases (e.g., polymerases from which modified recombinantpolymerases of the invention were derived, e.g., by mutation). Thepolymerases of the invention can also include any of the additionalfeatures for improved specificity, improved processivity, improvedretention time, improved surface stability, affinity tagging, and/or thelike noted herein.

These new polymerases are particularly well suited to DNA amplificationand/or sequencing applications, particularly sequencing protocols thatinclude detection in real time of the incorporation of labeled analogsinto DNA amplicons, since the increased phototolerance can prolonguseful life of the polymerase under assay conditions and the alteredrates, reduced or eliminated exonuclease activity, decreased branchfraction, improved complex stability, altered metal cofactorselectivity, or the like can facilitate discrimination of nucleotideincorporation events from non-incorporation events such as transientbinding of a mismatched nucleotide in the active site of the complex,improve processivity, and/or facilitate detection of incorporationevents.

Polymerases of the invention include, for example, a recombinantΦ29-type DNA polymerase that comprises a mutation at one or morepositions selected from the group consisting of Q99, K131, K132, K135,V141, L142, Y148, H149, Q183, G197, I201, K205, K206, Y224, D235, E239,V250, L253, C290, R306, R308, K311, E375, A437, T441, C455, K472, A484,I504, E508, D510, K512, L513, E515, D523, K536, and K539, whereidentification of positions is relative to wild-type Φ29 polymerase (SEQID NO:1). Optionally, the polymerase comprises mutations at two or more,three or more, four or more, five or more, or even six or more of thesepositions. For example, the polymerase can include a mutation atposition E375, a mutation at position K512, and a mutation at one ormore positions selected from the group consisting of Q99, K131, K132,K135, V141, L142, Y148, H149, Q183, G197, I201, K205, K206, Y224, D235,E239, V250, C290, R306, R308, K311, A437, T441, C455, K472, I504, E508,L513, E515, D523, K536, and K539 (where identification of positions isrelative to SEQ ID NO:1), and can optionally also include a mutation atone or more additional positions, e.g., as described herein. Similarly,for example, the polymerase can comprise mutations at positions 375,512, and 253, positions 375, 512, and 484, positions 253 and 484,positions 375, 512, 253, and 484, or positions 375, 512, 253, 484, and510, and a mutation at one or more positions selected from the groupconsisting of Q99, K131, K132, K135, V141, L142, Y148, H149, Q183, G197,I201, K205, K206, Y224, D235, E239, V250, C290, R306, R308, K311, A437,T441, C455, K472, I504, E508, L513, E515, D523, K536, and K539 (whereidentification of positions is relative to SEQ ID NO:1). A number ofexemplary substitutions at these (and other) positions are describedherein.

As a few examples, a mutation at E375 can comprise an amino acidsubstitution selected from the group consisting of E375Y, E375F, E375R,E375Q, E375H, E375L, E375A, E375K, E375S, E375T, E375C, E375G, andE375N; a mutation at position K512 can comprise an amino acidsubstitution selected from the group consisting of K512Y, K512F, K512I,K512M, K512C, K512E, K512G, K512H, K512N, K512Q, K512R, K512V, andK512H; a mutation at position L253 can comprise an amino acidsubstitution selected from the group consisting of L253A, L253H, L253S,and L253C; a mutation at position A484 can comprise an A484Esubstitution; and/or a mutation at position D510 can comprise a D510K orD510S substitution. Other exemplary substitutions include, e.g., Q99W,K131E, K131Q, K135Q, K135S, V141K, L142K, Y148I, H149D, Q183F, G197D,G197E, I201E, K205E, K205D, K205A, K206E, Y224K, D235E, E239G, V250A,V250I, C290F, R306Q, R308L, K311E, A437G, T441I, C455A, K472A, E508K,E515Q, E515P, E515K, and K536Q; additional substitutions are describedherein.

The polymerase mutations and mutational strategies noted herein can becombined with each other and with essentially any other availablemutations and mutational strategies to confer additional improvementsin, e.g., nucleotide analog specificity, enzyme processivity, improvedretention time of labeled nucleotides in polymerase-DNA-nucleotidecomplexes, phototolerance, and the like. For example, the mutations andmutational strategies herein can be combined with those taught in, e.g.,WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION byHanzel et al., WO 2008/051530 POLYMERASE ENZYMES AND REAGENTS FORENHANCED NUCLEIC ACID SEQUENCING by Rank et al., U.S. patent applicationpublication 2010-0075332 ENGINEERING POLYMERASES AND REACTION CONDITIONSFOR MODIFIED INCORPORATION PROPERTIES by Pranav Patel et al., U.S.patent application publication 2010-0093555 ENZYMES RESISTANT TOPHOTODAMAGE by Keith Bjornson et al., U.S. patent applicationpublication 2010-0112645 GENERATION OF MODIFIED POLYMERASES FOR IMPROVEDACCURACY 1N SINGLE MOLECULE SEQUENCING by Sonya Clark et al., U.S.patent application publication 2011-0189659 GENERATION OF MODIFIEDPOLYMERASES FOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCING by SonyaClark et al., and U.S. patent application publication 2012-0034602RECOMBINANT POLYMERASES FOR IMPROVED SINGLE MOLECULE SEQUENCING. Thiscombination of mutations/mutational strategies can be used to impartseveral simultaneous improvements to a polymerase (e.g., increasedphototolerance, decreased branch fraction formation, improvedspecificity, improved processivity, altered rates, improved retentiontime, improved stability of the closed complex, tolerance for aparticular metal cofactor, etc.). In addition, polymerases can befurther modified for application-specific reasons, such as to improveactivity of the enzyme when bound to a surface, as taught, e.g., in WO2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al. and WO2007/075873 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OFSURFACE ATTACHED PROTEINS by Hanzel et al., or to include purificationor handling tags as is taught in the cited references and as is commonin the art. Similarly, the modified polymerases described herein can beemployed in combination with other strategies to improve polymeraseperformance, for example, reaction conditions for controlling polymeraserate constants such as taught in U.S. patent application publicationU.S. 2009-0286245 entitled “Two slow-step polymerase enzyme systems andmethods.”

Also taught are approaches for modifying polymerases to enhance one ormore properties exhibited by the polymerases or to confer an additionalproperty not provided by a starting combination of mutations. Forexample, provided below are approaches for structure-based design ofpolymerases with increased resistance to photodamage (increasedphototolerance).

DNA Polymerases

DNA polymerases that can be modified to have increased phototoleranceand/or other desirable properties as described herein are generallyavailable. DNA polymerases are sometimes classified into six main groupsbased upon various phylogenetic relationships, e.g., with E. coli Pol I(class A), E. coli Pol II (class B), E. coli Pol III (class C),Euryarchaeotic Pol II (class D), human Pol beta (class X), and E. coliUmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variant (class Y).For a review of recent nomenclature, see, e.g., Burgers et al. (2001)“Eukaryotic DNA polymerases: proposal for a revised nomenclature” JBiol. Chem. 276(47):43487-90. For a review of polymerases, see, e.g.,Hübscher et al. (2002) “Eukaryotic DNA Polymerases” Annual Review ofBiochemistry Vol. 71: 133-163; Alba (2001) “Protein Family Review:Replicative DNA Polymerases” Genome Biology 2(1):reviews 3002.1-3002.4;and Steitz (1999) “DNA polymerases: structural diversity and commonmechanisms” J Biol Chem 274:17395-17398. The basic mechanisms of actionfor many polymerases have been determined. The sequences of literallyhundreds of polymerases are publicly available, and the crystalstructures for many of these have been determined or can be inferredbased upon similarity to solved crystal structures for homologouspolymerases. For example, the crystal structure of Φ29, a preferred typeof parental enzyme to be modified according to the invention, isavailable.

Many such polymerases that are suitable for modification are available,e.g., for use in sequencing, labeling, and amplification technologies.For example, human DNA Polymerase Beta is available from R&D systems.DNA polymerase I is available from Epicenter, GE Health Care,Invitrogen, New England Biolabs, Promega, Roche Applied Science, SigmaAldrich, and many others. The Klenow fragment of DNA Polymerase I isavailable in both recombinant and protease digested versions, from,e.g., Ambion, Chimerx, eEnzyme LLC, GE Health Care, Invitrogen, NewEngland Biolabs, Promega, Roche Applied Science, Sigma Aldrich and manyothers. Φ29 DNA polymerase is available from e.g., Epicentre. Poly Apolymerase, reverse transcriptase, Sequenase, SP6 DNA polymerase, T4 DNApolymerase, T7 DNA polymerase, and a variety of thermostable DNApolymerases (Taq, hot start, titanium Taq, etc.) are available from avariety of these and other sources. Recent commercial DNA polymerasesinclude Phusion™ High-Fidelity DNA Polymerase, available from NewEngland Biolabs; GoTaq® Flexi DNA Polymerase, available from Promega;RepliPHI™ Φ29 DNA Polymerase, available from Epicentre Biotechnologies;PfuUltra™ Hotstart DNA Polymerase, available from Stratagene; KOD HiFiDNA Polymerase, available from Novagen; and many others.Biocompare(dot)com provides comparisons of many different commerciallyavailable polymerases.

DNA polymerases that are preferred substrates for mutation to increasephototolerance, reduce reaction rates, reduce or eliminate exonucleaseactivity, decrease branching fraction, improve closed complex stability,alter metal cofactor selectivity, and/or alter one or more otherproperty described herein include Taq polymerases, exonuclease deficientTaq polymerases, E. coli DNA Polymerase 1, Klenow fragment, reversetranscriptases, Φ29 related polymerases including wild type Φ29polymerase and derivatives of such polymerases such as exonucleasedeficient forms, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase,etc.

In one aspect, the polymerase that is modified is a Φ29-type DNApolymerase. For example, the modified recombinant DNA polymerase can behomologous to a wild-type or exonuclease deficient Φ29 DNA polymerase,e.g., as described in U.S. Pat. Nos. 5,001,050, 5,198,543, or 5,576,204.Alternately, the modified recombinant DNA polymerase can be homologousto another Φ29-type DNA polymerase, such as B103, GA-1, PZA, Φ15, BS32,M2Y (also known as M2), Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4,PR5, PR722, L17, AV-1, Φ21, or the like. For nomenclature, see also,Meijer et al. (2001) “Φ29 Family of Phages” Microbiology and MolecularBiology Reviews, 65(2):261-287. See, e.g., SEQ ID NO:1 for the aminoacid sequence of wild-type Φ29 polymerase, SEQ ID NO:2 for the aminoacid sequence of wild-type M2Y polymerase, SEQ ID NO:3 for the aminoacid sequence of wild-type B103 polymerase, SEQ ID NO:4 for the aminoacid sequence of wild-type GA-1 polymerase, SEQ ID NO:5 for the aminoacid sequence of wild-type AV-1 polymerase, and SEQ ID NO:6 for theamino acid sequence of wild-type CP-1 polymerase.

In addition to wild-type polymerases, chimeric polymerases made from amosaic of different sources can be used. For example, Φ29-typepolymerases made by taking sequences from more than one parentalpolymerase into account can be used as a starting point for mutation toproduce the polymerases of the invention. Chimeras can be produced,e.g., using consideration of similarity regions between the polymerasesto define consensus sequences that are used in the chimera, or usinggene shuffling technologies in which multiple Φ29-related polymerasesare randomly or semi-randomly shuffled via available gene shufflingtechniques (e.g., via “family gene shuffling”; see Crameri et al. (1998)“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution” Nature 391:288-291; Clackson et al. (1991) “Makingantibody fragments using phage display libraries” Nature 352:624-628;Gibbs et al. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): amethod for enhancing the frequency of recombination with familyshuffling” Gene 271:13-20; and Hiraga and Arnold (2003) “General methodfor sequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296). In these methods, the recombination points can bepredetermined such that the gene fragments assemble in the correctorder. However, the combinations, e.g., chimeras, can be formed atrandom. For example, using methods described in Clarkson et al., fivegene chimeras, e.g., comprising segments of a Phi29 polymerase, a PZApolymerase, a M2 polymerase, a B103 polymerase, and a GA-1 polymerase,can be generated. Appropriate mutations to increase phototoleranceand/or alter another desirable property as described herein can beintroduced into the chimeras.

Available DNA polymerase enzymes have also been modified in any of avariety of ways, e.g., to reduce or eliminate exonuclease activities(many native DNA polymerases have a proof-reading exonuclease functionthat interferes with, e.g., sequencing applications), to simplifyproduction by making protease digested enzyme fragments such as theKlenow fragment recombinant, etc. As noted, polymerases have also beenmodified to confer improvements in specificity, processivity, andretention time of labeled nucleotides in polymerase-DNA-nucleotidecomplexes (e.g., WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUEINCORPORATION by Hanzel et al. and WO 2008/051530 POLYMERASE ENZYMES ANDREAGENTS FOR ENHANCED NUCLEIC ACID SEQUENCING by Rank et al.), to alterbranching fraction and translocation (e.g., U.S. patent applicationpublication 2010-0075332 by Pranav Patel et al. entitled “ENGINEERINGPOLYMERASES AND REACTION CONDITIONS FOR MODIFIED INCORPORATIONPROPERTIES”), to increase photostability (e.g., U.S. patent applicationpublication 2010-0093555 ENZYMES RESISTANT TO PHOTODAMAGE by KeithBjornson et al.), to slow one or more catalytic steps during thepolymerase kinetic cycle, increase closed complex stability, decreasebranching fraction, alter cofactor selectivity, and increase yield,thermostability, accuracy, speed, and readlength (e.g., U.S. patentapplication publication 2010-0112645 GENERATION OF MODIFIED POLYMERASESFOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCING by Sonya Clark etal., U.S. patent application publication 2011-0189659 GENERATION OFMODIFIED POLYMERASES FOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCINGby Sonya Clark et al., and U.S. patent application publication2012-0034602 RECOMBINANT POLYMERASES FOR IMPROVED SINGLE MOLECULESEQUENCING), and to improve surface-immobilized enzyme activities (e.g.,WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al. andWO 2007/075873 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OFSURFACE ATTACHED PROTEINS by Hanzel et al.). Any of these availablepolymerases can be modified in accordance with the invention.

Nucleotide Analogs

As discussed, various polymerases of the invention can incorporate oneor more nucleotide analogs into a growing oligonucleotide chain. Uponincorporation, the analog can leave a residue that is the same as ordifferent than a natural nucleotide in the growing oligonucleotide (thepolymerase can incorporate any non-standard moiety of the analog, or cancleave it off during incorporation into the oligonucleotide). A“nucleotide analog” herein is a compound, that, in a particularapplication, functions in a manner similar or analogous to a naturallyoccurring nucleoside triphosphate (a “nucleotide”), and does nototherwise denote any particular structure. A nucleotide analog is ananalog other than a standard naturally occurring nucleotide, i.e., otherthan A, G, C, T, or U, though upon incorporation into theoligonucleotide, the resulting residue in the oligonucleotide can be thesame as (or different from) an A, G, C, T, or U residue.

In one useful aspect of the invention, nucleotide analogs can also bemodified to achieve any of the improved properties desired. For example,various linkers or other substituents can be incorporated into analogsthat have the effect of reducing branching fraction, improvingprocessivity, or altering rates. Modifications to the analogs caninclude extending the phosphate chains, e.g., to include a tetra-,penta-, hexa- or heptaphosphate group, and/or adding chemical linkers toextend the distance between the nucleotide base and the dye molecule,e.g., a fluorescent dye molecule. Substitution of one or morenon-bridging oxygen in the polyphosphate, for example with S or BH₃, canchange the polymerase reaction kinetics, e.g., to achieve a systemhaving two slow steps as described hereinbelow. Optionally, one or more,two or more, three or more, or four or more non-bridging oxygen atoms inthe polyphosphate group of the analog has an S substituted for an O.While not being bound by theory, it is believed that the properties ofthe nucleotide, such as the metal chelation properties,electronegativity, or steric properties, can be altered by substitutionof the non-bridging oxygen(s).

Many nucleotide analogs are available and can be incorporated by thepolymerases of the invention. These include analog structures with coresimilarity to naturally occurring nucleotides, such as those thatcomprise one or more substituent on a phosphate, sugar, or base moietyof the nucleoside or nucleotide relative to a naturally occurringnucleoside or nucleotide. In one embodiment, the nucleotide analogincludes three phosphate containing groups; for example, the analog canbe a labeled nucleoside triphosphate analog and/or an α-thiophosphatenucleotide analog having three phosphate groups. In one embodiment, anucleotide analog can include one or more extra phosphate containinggroups, relative to a nucleoside triphosphate. For example, a variety ofnucleotide analogs that comprise, e.g., from 4-6 or more phosphates aredescribed in detail in U.S. patent application publication 2007-0072196,incorporated herein by reference in its entirety for all purposes. Otherexemplary useful analogs, including tetraphosphate and pentaphosphateanalogs, are described in U.S. Pat. No. 7,041,812, incorporated hereinby reference in its entirety for all purposes.

For example, the analog can include a labeled compound of the formula:

-   wherein B is a nucleobase (and optionally includes a label); S is    selected from a sugar moiety, an acyclic moiety or a carbocyclic    moiety (and optionally includes a label); L is an optional    detectable label; R₁ is selected from O and S; R₂, R₃ and R₄ are    independently selected from O, NH, S, methylene, substituted    methylene, C(O), C(CH₂), CNH₂, CH₂CH₂, C(OH)CH₂R where R is    4-pyridine or 1-imidazole, provided that R₄ may additionally be    selected from

-    R₅, R₆, R₇, R₈, R₁₁ and R₁₃ are, when present, each independently    selected from O, BH₃, and S; and R₉, R₁₀ and R₁₂ are independently    selected from O, NH, S, methylene, substituted methylene, CNH₂,    CH₂CH₂, and C(OH)CH₂R where R is 4-pyridine or 1-imidazole. In some    cases, phosphonate analogs may be employed as the analogs, e.g.,    where one of R₂, R₃, R₄, R₉, R₁₀ or R₁₂ are not O, e.g., they are    methyl etc. See, e.g., U.S. patent application publication    2007-0072196, previously incorporated herein by reference in its    entirety for all purposes.

The base moiety incorporated into the analog is generally selected fromany of the natural or non-natural nucleobases or nucleobase analogs,including, e.g., purine or pyrimidine bases that are routinely found innucleic acids and available nucleic acid analogs, including adenine,thymine, guanine, cytidine, uracil, and in some cases, inosine. Asnoted, the base optionally includes a label moiety. For convenience,nucleotides and nucleotide analogs are generally referred to based upontheir relative analogy to naturally occurring nucleotides. As such, ananalog that operates, functionally, like adenosine triphosphate may begenerally referred to herein by the shorthand letter A. Likewise, thestandard abbreviations of T, G, C, U and I may be used in referring toanalogs of naturally occurring nucleosides and nucleotides typicallyabbreviated in the same fashion. In some cases, a base may function in amore universal fashion, e.g., functioning like any of the purine basesin being able to hybridize with any pyrimidine base, or vice versa. Thebase moieties used in the present invention may include the conventionalbases described herein or they may include such bases substituted at oneor more side groups, or other fluorescent bases or base analogs, such as1,N6 ethenoadenosine or pyrrolo C, in which an additional ring structurerenders the B group neither a purine nor a pyrimidine. For example, incertain cases, it may be desirable to substitute one or more side groupsof the base moiety with a labeling group or a component of a labelinggroup, such as one of a donor or acceptor fluorophore, or other labelinggroup. Examples of labeled nucleobases and processes for labeling suchgroups are described in, e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928,each of which is incorporated herein by reference in its entirety forall purposes.

In the analogs, the S group is optionally a sugar moiety that provides asuitable backbone for a synthesizing nucleic acid strand. For example,the sugar moiety is optionally selected from a D-ribosyl, 2′ or 3′D-deoxyribosyl, 2′,3′-D-dideoxyribosyl, 2′,3′-D-didehydrodideoxyribosyl, 2′ or 3′ alkoxyribosyl, 2′ or 3′ aminoribosyl, 2′ or 3′ mercaptoribosyl, 2′ or 3′ alkothioribosyl,acyclic, carbocyclic or other modified sugar moieties. A variety ofcarbocyclic or acyclic moieties can be incorporated as the “S” group inplace of a sugar moiety, including, e.g., those described in U.S. PatentApplication Publication No. 2003/0124576, which is incorporated hereinby reference in its entirety for all purposes.

For most cases, the phosphorus containing chain in the analogs, e.g., atriphosphate in conventional NTPs, is preferably coupled to the 5′hydroxyl group, as in natural nucleoside triphosphates. However, in somecases, the phosphorus containing chain is linked to the S group by the3′ hydroxyl group.

L generally refers to a detectable labeling group that is coupled to theterminal phosphorus atom via the R₄ (or R₁₀ or R₁₂ etc.) group. Thelabeling groups employed in the analogs of the invention may compriseany of a variety of detectable labels. Detectable labels generallydenote a chemical moiety that provides a basis for detection of theanalog compound separate and apart from the same compound lacking such alabeling group. Examples of labels include, e.g., optical labels, e.g.,labels that impart a detectable optical property to the analog,electrochemical labels, e.g., labels that impart a detectable electricalor electrochemical property to the analog, and physical labels, e.g.,labels that impart a different physical or spatial property to theanalog, e.g., a mass tag or molecular volume tag. In some casesindividual labels or combinations may be used that impart more than oneof the aforementioned properties to the analogs of the invention.

Optionally, the labeling groups incorporated into the analogs compriseoptically detectable moieties, such as luminescent, chemiluminescent,fluorescent, fluorogenic, chromophoric and/or chromogenic moieties, withfluorescent and/or fluorogenic labels being preferred. A variety ofdifferent label moieties are readily employed in nucleotide analogs.Such groups include, e.g., fluorescein labels, rhodamine labels, cyaninelabels (i.e., Cy3, Cy5, and the like, generally available from theAmersham Biosciences division of GE Healthcare), the Alexa family offluorescent dyes and other fluorescent and fluorogenic dyes availablefrom Molecular Probes/Invitrogen, Inc. and described in ‘The Handbook—AGuide to Fluorescent Probes and Labeling Technologies, Eleventh Edition’(2010) (available from Invitrogen, Inc./Molecular Probes). A variety ofother fluorescent and fluorogenic labels for use with nucleosidepolyphosphates, and which would be applicable to the nucleotide analogsincorporated by the polymerases of the present invention, are describedin, e.g., U.S. Patent Application Publication No. 2003/0124576,previously incorporated herein by reference in its entirety for allpurposes.

Additional details regarding labels, analogs, and methods of making suchanalogs can be found in U.S. patent application publication2007-0072196, WO 2007/041342 Labeled Nucleotide Analogs and UsesTherefor, WO 2009/114182 Labeled Reactants and Their Uses, U.S. patentapplication publication 2009-0208957 Alternate Labelling Strategies forSingle Molecule Sequencing, U.S. patent application Ser. No. 13/218,412Functionalized Cyanine Dyes, U.S. patent application Ser. No. 13/218,395Functionalized Cyanine Dyes, U.S. patent application Ser. No. 13/218,428Cyanine Dyes, and U.S. patent application Ser. No. 13/218,382Scaffold-Based Polymerase Enzyme Substrates, each of which isincorporated herein by reference in its entirety for all purposes.

Thus, in one illustrative example, the analog can be a phosphate analog(e.g., an analog that has more than the typical number of phosphatesfound in nucleoside triphosphates) that includes, e.g., an Alexa dyelabel. For example, an Alexa488 dye can be labeled on a delta phosphateof a tetraphosphate analog (denoted, e.g., A488dC4P or A488dA4P, shownin FIG. 2, for the Alexa488 labeled tetraphosphate analogs of C and A,respectively), or an Alexa568 or Alexa633 dye can be used (e.g.,A568dC4P and A633dC4P, respectively, for labeled tetraphosphate analogsof C or A568dT6P for a labeled hexaphosphate analog of T), or anAlexa546 dye can be used (e.g., A546dG4P), or an Alexa594 dye can beused (e.g., A594dT4P). As additional examples, an Alexa555 dye (e.g.,A555dC6P or A555dA6P), an Alexa 647 dye (e.g., A647dG6P), an Alexa 568dye (e.g., A568dT6P), and/or an Alexa660 dye (e.g., A660dA6P orA660dC6P) can be used in, e.g., single molecule sequencing. Similarly,to facilitate color separation, a pair of fluorophores exhibiting FRET(fluorescence resonance energy transfer) can be labeled on a deltaphosphate of a tetraphosphate analog (denoted, e.g., FAM-amb-A532dG4P orFAM-amb-A594dT4P).

Applications for Enhanced Nucleic Acid Amplification and Sequencing

Polymerases of the invention, e.g., modified recombinant polymerases,are optionally used in combination with nucleotides and/or nucleotideanalogs and nucleic acid templates (e.g., DNA, RNA, or hybrids, analogs,derivatives, or mimetics thereof) to copy the template nucleic acid.That is, a mixture of the polymerase, nucleotides/analogs, andoptionally other appropriate reagents, the template and a replicationinitiating moiety (e.g., primer) is reacted such that the polymerasesynthesizes nucleic acid (e.g., extends the primer) in atemplate-dependent manner. The replication initiating moiety can be astandard oligonucleotide primer, or, alternatively, a component of thetemplate, e.g., the template can be a self-priming single stranded DNA,a nicked double stranded DNA, or the like. Similarly, a terminal proteincan serve as an initiating moiety. At least one nucleotide analog can beincorporated into the DNA. The template DNA can be a linear or circularDNA, and in certain applications, is desirably a circular template(e.g., for rolling circle replication or for sequencing of circulartemplates). Optionally, the composition can be present in an automatedDNA replication and/or sequencing system.

Incorporation of labeled nucleotide analogs by the polymerases of theinvention is particularly useful in a variety of different nucleic acidanalyses, including real-time monitoring of DNA polymerization. Thelabel can itself be incorporated, or more preferably, can be releasedduring incorporation of the analog. For example, analog incorporationcan be monitored in real-time by monitoring label release duringincorporation of the analog by the polymerase. The portion of the analogthat is incorporated can be the same as a natural nucleotide, or caninclude features of the analog that differ from a natural nucleotide.

In general, label incorporation or release can be used to indicate thepresence and composition of a growing nucleic acid strand, e.g.,providing evidence of template replication/amplification and/or sequenceof the template. Signaling from the incorporation can be the result ofdetecting labeling groups that are liberated from the incorporatedanalog, e.g., in a solid phase assay, or can arise upon theincorporation reaction. For example, in the case of FRET labels where abound label is quenched and a free label is not, release of a labelgroup from the incorporated analog can give rise to a fluorescentsignal. Alternatively, the enzyme may be labeled with one member of aFRET pair proximal to the active site, and incorporation of an analogbearing the other member will allow energy transfer upon incorporation.The use of enzyme bound FRET components in nucleic acid sequencingapplications is described, e.g., in U.S. Patent Application PublicationNo. 2003/0044781, incorporated herein by reference.

In one example reaction of interest, a polymerase reaction can beisolated within an extremely small observation volume that effectivelyresults in observation of individual polymerase molecules. As a result,the incorporation event provides observation of an incorporatingnucleotide analog that is readily distinguishable from non-incorporatednucleotide analogs. In a preferred aspect, such small observationvolumes are provided by immobilizing the polymerase enzyme within anoptical confinement, such as a Zero Mode Waveguide (ZMW). For adescription of ZMWs and their application in single molecule analyses,and particularly nucleic acid sequencing, see, e.g., U.S. PatentApplication Publication No. 2003/0044781 and U.S. Pat. No. 6,917,726,each of which is incorporated herein by reference in its entirety forall purposes. See also Levene et al. (2003) “Zero-mode waveguides forsingle-molecule analysis at high concentrations” Science 299:682-686,Eid et al. (2009) “Real-time DNA sequencing from single polymerasemolecules” Science 323:133-138, and U.S. Pat. Nos. 7,056,676, 7,056,661,7,052,847, and 7,033,764, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes.

In general, a polymerase enzyme is complexed with the template strand inthe presence of one or more nucleotides and/or one or more nucleotideanalogs. For example, in certain embodiments, labeled analogs arepresent representing analogous compounds to each of the four naturalnucleotides, A, T, G and C, e.g., in separate polymerase reactions, asin classical Sanger sequencing, or multiplexed together, e.g., in asingle reaction, as in multiplexed sequencing approaches. When aparticular base in the template strand is encountered by the polymeraseduring the polymerization reaction, it complexes with an availableanalog that is complementary to such nucleotide, and incorporates thatanalog into the nascent and growing nucleic acid strand. In one aspect,incorporation can result in a label being released, e.g., inpolyphosphate analogs, cleaving between the α and β phosphorus atoms inthe analog, and consequently releasing the labeling group (or a portionthereof). The incorporation event is detected, either by virtue of alonger presence of the analog and, thus, the label, in the complex, orby virtue of release of the label group into the surrounding medium.Where different labeling groups are used for each of the types ofanalogs, e.g., A, T, G or C, identification of a label of anincorporated analog allows identification of that analog andconsequently, determination of the complementary nucleotide in thetemplate strand being processed at that time. Sequential reaction andmonitoring permits real-time monitoring of the polymerization reactionand determination of the sequence of the template nucleic acid. As notedabove, in particularly preferred aspects, the polymerase enzyme/templatecomplex is provided immobilized within an optical confinement thatpermits observation of an individual complex, e.g., a zero modewaveguide. For additional information on single molecule sequencingmonitoring incorporation of phosphate-labeled analogs in real time, see,e.g., Eid et al. (2009) “Real-time DNA sequencing from single polymerasemolecules” Science 323:133-138.

In a first exemplary technique, as schematically illustrated in FIG. 3Panel A, a nucleic acid synthesis complex, including a polymerase enzyme202, a template sequence 204 and a complementary primer sequence 206, isprovided immobilized within an observation region 200 that permitsillumination (as shown by hv) and observation of a small volume thatincludes the complex without excessive illumination of the surroundingvolume (as illustrated by dashed line 208). By illuminating andobserving only the volume immediately surrounding the complex, one canreadily identify fluorescently labeled nucleotides that becomeincorporated during that synthesis, as such nucleotides are retainedwithin that observation volume by the polymerase for longer periods thanthose nucleotides that are simply randomly diffusing into and out ofthat volume.

In particular, as shown in Panel B of FIG. 3, when a nucleotide, e.g.,A, is incorporated into DNA by the polymerase, it is retained within theobservation volume for a prolonged period of time, and upon continuedillumination yields a prolonged fluorescent signal (shown by peak 210).By comparison, randomly diffusing and not incorporated nucleotidesremain within the observation volume for much shorter periods of time,and thus produce only transient signals (such as peak 212), many ofwhich go undetected due to their extremely short duration.

In particularly preferred exemplary systems, the confined illuminationvolume is provided through the use of arrays of optically confinedapertures termed zero mode waveguides (ZMWs), e.g., as shown by confinedreaction region 200 (see, e.g., U.S. Pat. No. 6,917,726, which isincorporated herein by reference in its entirety for all purposes). Forsequencing applications, the DNA polymerase is typically providedimmobilized upon the bottom of the ZMW, although another component ofthe complex (e.g., a primer or template) is optionally immobilized onthe bottom of the ZMW to localize the complex. See, e.g., Korlach et al.(2008) PNAS U.S.A. 105(4):1176-1181 and U.S. patent applicationpublication 2008-0032301, each of which is incorporated herein byreference in its entirety for all purposes.

In operation, the fluorescently labeled nucleotides (shown as A, C, Gand T) bear one or more fluorescent dye groups on a terminal phosphatemoiety that is cleaved from the nucleotide upon incorporation. As aresult, synthesized nucleic acids do not bear the build-up offluorescent labels, as the labeled polyphosphate groups diffuse awayfrom the complex following incorporation of the associated nucleotide,nor do such labels interfere with the incorporation event. See, e.g.,Korlach et al. (2008) Nucleosides, Nucleotides and Nucleic Acids27:1072-1083.

A fluorescence time trace for a ZMW, showing pulses (peaks) representingincorporation of different nucleotide analogs, is presented in FIG. 4. Apulse width and interpulse distance are illustrated on the trace. Theinset schematically illustrates the catalytic cycle forpolymerase-mediated nucleic acid primer extension according to theexemplary reaction scheme described in U.S. patent applicationpublication 2012-0034602; the box indicates the portion of the catalyticcycle that corresponds to the pulse when sequencing is performed withphosphate-labeled nucleotide analogs. The remainder of the cyclecorresponds to the interpulse distance.

In a second exemplary technique, the immobilized complex and thenucleotides to be incorporated are each provided with interactivelabeling components. Upon incorporation, the nucleotide borne labelingcomponent is brought into sufficient proximity to the complex borne (orcomplex proximal) labeling component, such that these components producea characteristic signal event. For example, the polymerase may beprovided with a fluorophore that provides fluorescent resonant energytransfer (FRET) to appropriate acceptor fluorophores. These acceptorfluorophores are provided upon the nucleotide to be incorporated, whereeach type of nucleotide bears a different acceptor fluorophore, e.g.,that provides a different fluorescent signal. Upon incorporation, thedonor and acceptor are brought close enough together to generate energytransfer signal. By providing different acceptor labels on the differenttypes of nucleotides, one obtains a characteristic FRET-basedfluorescent signal for the incorporation of each type of nucleotide, asthe incorporation is occurring.

In a related aspect, a nucleotide analog may include two interactingfluorophores that operate as a donor/quencher pair, where one member ispresent on the nucleobase or other retained portion of the nucleotide,while the other member is present on a phosphate group or other portionof the nucleotide that is released upon incorporation, e.g., a terminalphosphate group. Prior to incorporation, the donor and quencher aresufficiently proximal on the same analog as to provide characteristicsignal quenching. Upon incorporation and cleavage of the terminalphosphate groups, e.g., bearing a donor fluorophore, the quenching isremoved and the resulting characteristic fluorescent signal of the donoris observable.

In exploiting the foregoing processes, where the incorporation reactionoccurs too rapidly, it may result in the incorporation event not beingdetected, i.e., the event speed exceeds the detection speed of themonitoring system. The missed detection of incorporated nucleotides canlead to an increased rate of errors in sequence determination, asomissions in the real sequence. In order to mitigate the potential formissed pulses due to short reaction or product release times, in oneaspect, the current invention can result in increased reaction and/orproduct release times during incorporation cycles. Similarly, very shortinterpulse distances can occasionally cause pulse merging. An advantageof employing polymerases with reduced reaction rates, e.g., polymerasesexhibiting decreased rates and/or two slow-step kinetics as described inU.S. patent application publications 2009-0286245 and 2010-0112645, isan increased frequency of longer, detectable, binding events. Thisadvantage may also be seen as an increased ratio of longer, detectablepulses to shorter, non-detectable pulses, where the pulses representbinding events.

In addition to their use in sequencing, the polymerases of the inventionare also useful in a variety of other genotyping analyses, e.g., SNPgenotyping using single base extension methods, real time monitoring ofamplification, e.g., RT-PCR methods, and the like. The polymerases ofthe invention are also useful in amplifying nucleic acids, e.g., DNAs orRNAs, including, for example, in applications such as whole genomeamplification. For example, polymerases of the invention that showincreased thermostability or resistance to organic solvents (e.g.,DMSO), or that otherwise exhibit an improved ability to read throughdamaged, modified, or other “difficult” stretches of nucleic acidtemplate, can be suitably employed in whole genome amplification. Forreview of whole genome amplification, see, e.g., Silander and Saarela(2008) “Whole Genome Amplification with Phi29 DNA Polymerase to EnableGenetic or Genomic Analysis of Samples of Low DNA Yield” Methods inMolecular Biology 439:1-18 and Pinard et al. (2006) “Assessment of wholegenome amplification-induced bias through high-throughput, massivelyparallel whole genome sequencing” BMC Genomics 7:216. Further detailsregarding sequencing and nucleic acid amplification can be found, e.g.,in Sambrook, Ausubel, and Innis, all infra.

Recombinant Polymerases with Increased Phototolerance

The compositions of the invention comprise a modified recombinant DNApolymerase which exhibits one or more altered properties desirable insingle molecule sequencing applications or other applications involvingnucleic acid synthesis. An exemplary property of certain polymerases ofthe invention is increased phototolerance relative to a wild-type orparental polymerase. Other exemplary properties include altered kineticbehavior (e.g., demonstration of slow catalytic steps), exonucleasedeficiency, increased closed complex stability, altered (e.g., reduced)branching fraction, altered cofactor selectivity, increased yield,increased thermostability, increased accuracy, increased speed, andincreased readlength.

As will be understood, polymerases of the invention can display one ofthe aforementioned properties alone or can display two or more of theproperties in combination. Moreover, it will be understood that while apolymerase or group of polymerases may be described with respect to aparticular property, the polymerase(s) may possess additional modifiedproperties not mentioned in every instance for ease of discussion. Itwill also be understood that particular properties are observed undercertain conditions. For example, a photoprotective mutation can, e.g.,confer increased readlength (as compared to a parental polymeraselacking the mutation) when observed with an excitation light source at aconstant power or it can confer increased accuracy at a higher power. Asingle mutation (e.g., a single amino acid substitution, deletion,insertion, or the like) may give rise to the one or more alteredproperties, or the one or more properties may result from two or moremutations which act in concert to confer the desired activity. Therecombinant polymerases, mutations, and altered properties exhibited bythe recombinant polymerases are set forth in greater detail below.

Detection of optical labels in an enzymatic reaction generally entailsdirecting excitation radiation at the reaction mixture to excite alabeling group present in the mixture, which is then separatelydetectable. However, prolonged exposure of chemical and biochemicalreactants to radiation (e.g., light) energy during the excitation anddetection of optical labels can damage components of the reactionmixture, e.g., enzymes, proteins, substrates, or the like. For example,it has been observed that, in template-directed synthesis of nucleicacids from fluorescently labeled nucleotides or nucleotide analogs,sustained exposure of the DNA polymerase to excitation radiation used inthe detection of the relevant label (e.g., fluorophore) reduces theenzyme's processivity and polymerase activity. Although illuminatedreactions typically proceed under conditions where the reactants (e.g.,enzyme molecules, etc.) are present in excess such that any adverseeffects of photodamage on any single enzyme molecule in the reaction mixdo not, in general, affect operation of the assay, an increasing numberof analyses that entail the use of optical labels are performed withreactants at very low concentrations. For example, polymerases can beused to synthesize DNAs from fluorescently labeled nucleotide analogs inmicrofluidic or nanofluidic reaction vessels or channels or in opticallyconfined reaction volumes, e.g., in a zero-mode waveguide (ZMW) or ZMWarray as described above. Analysis of small, single-analyte reactionvolumes is becoming increasingly important in high-throughputapplications, e.g., in DNA sequencing. However, in such reactant-limitedanalyses, any degradation of a critical reagent such as an enzymemolecule due to photodamage can dramatically interfere with theanalysis.

Polymerases that exhibit decreased sensitivity to photodamage (increasedphototolerance) are thus desirable for use in a variety of single- orlow-number enzyme analyses, including, but not limited to, DNAsequencing (e.g., single molecule sequencing), nucleic acidamplification, and others. Exemplary approaches to producing polymeraseswith increased resistance to photodamage by, e.g., replacing residuessusceptible to oxidative damage have been described in U.S. patentapplication publication 2010-0093555. Additional approaches aredescribed below.

Without limitation to any particular mechanism, observation ofpolymerase performance in single molecule sequencing reactions usinglabeled nucleotide analogs has revealed that, in many instances,photodamage involves collision of the dye moiety of an analog with thepolymerase followed by crosslink formation between the dye and thepolymerase. A novel approach to increasing polymerase phototolerancethus involves reducing the frequency of such collisions. In oneapproach, again without limitation to any particular mechanism, sincethe nucleotide analog is negatively charged, the frequency of collisionsbetween the polymerase and the label is reduced by introducing negativecharges to and/or removing positive charges from the surface of thepolymerase that is within reach of the dye moiety. An electrostaticsurface representation of Φ29 polymerase with a bound analog is shown inFIG. 5 (dark gray is positively charged surface, and medium gray isnegatively charged). It will be evident that residues playing anessential role in nucleotide binding and/or catalysis are not preferredsites for substitution. Exemplary locations where positive surfacecharge can be reduced without loss of performance in sequencingreactions are shown in dark gray in FIG. 6.

Positions of particular interest include, e.g., K131, K132, K135, H149,G197, I201, K205, K206, K472, K536, and K539, where positions areidentified relative to wild-type Φ29 polymerase (SEQ ID NO:1). Toproduce a polymerase with increased phototolerance, a residue at one (ormore) of these positions can be substituted with another residue,preferably a non-positively charged residue (e.g., a negatively chargedresidue such as Asp or Glu, or an uncharged polar residue, e.g., Asn,Gln, or Ser). Suitable substitutions at these positions include, forexample, K131E, K131Q, K131S, K131D, K131A, K131H, K131L, K131Y, K131I,K131N, K131C, K131F, K131G, K131P, K131R, K131T, K131V, K131W, K135Q,K135S, K135N, H149D, G197D, G197E, I201E, K205E, K205D, K205A, K206E,K472A, K536Q, and K539Q.

Decreasing the overall positive surface charge on the polymerase in thevicinity of the analog binding pocket can, however, affect binding rateof the analog, resulting in undesirably lengthened interpulse distancesor the like. Increasing positive charge in areas of the polymerase'ssurface that are less accessible to the dye moiety can compensate forthis effect by narrowing interpulse distances and increasing polymerasespeed. (It will be evident that such mutations can be employed toincrease speed regardless of the presence or absence of mutations thatdecrease speed while increasing phototolerance.)

Positions of particular interest include, e.g., V141, L142, I504, E508,D510, L513, and D523, where positions are identified relative towild-type Φ29 polymerase (SEQ ID NO:1). To increase the overall positivesurface charge, a residue at one or more of these positions can besubstituted with another residue, e.g., with an uncharged residue wherethe residue was originally negatively charged, or more preferably, witha positively charged residue (e.g., Lys, H is, or Arg). Suitablesubstitutions at these positions include, for example, V141K, L142K,E508K, D510S, and D510K.

As will be appreciated, recombinant polymerases that exhibit increasedphototolerance and/or speed can also include additional mutations (e.g.,amino acid substitutions, deletions, insertions, exogenous features atthe N- and/or C-terminus, and/or the like) which confer one or moreadditional desirable properties, e.g., reduced or eliminated exonucleaseactivity, convenient surface immobilization, increased closed complexstability, reduced or increased branching, selectivity for particularmetal cofactors, increased yield, increased thermostability, increasedaccuracy, increased speed, and/or increased readlength.

Design and Characterization of Recombinant Polymerases

In addition to methods of using the polymerases and other compositionsherein, the present invention also includes methods of making thepolymerases. (Polymerases made by the methods are also a feature of theinvention, and it will be evident that, although various designstrategies are detailed herein, no limitation of the resultingpolymerases to any particular mechanism is thereby intended.) Asdescribed, methods of making a recombinant DNA polymerase can includestructurally modeling a parental polymerase, e.g., using any availablecrystal structure and molecular modeling software or system. Based onthe modeling, one or more amino acid residue positions in the polymeraseare identified as targets for mutation. For example, one or more featureaffecting phototolerance, closed complex stability, nucleotide access toor removal from the active site (and, thereby, branching), binding of aDNA or nucleotide analog, product binding, etc. is identified. Theseresidues can be, e.g., in the active site or a binding pocket or in adomain such as the exonuclease, TPR2 or thumb domain (or interfacebetween domains) or proximal to such domains. The DNA polymerase ismutated to include different residues at such positions (e.g., anotherone of the nineteen other commonly occurring natural amino acids or anon-natural amino acid, e.g., a nonpolar and/or aliphatic residue, apolar uncharged residue, an aromatic residue, a positively chargedresidue, or a negatively charged residue), and then screened for anactivity of interest (e.g., phototolerance, processivity, k_(off),K_(d), branching fraction, decreased rate constant, balanced rateconstants, accuracy, speed, thermostability, yield, cofactorselectivity, etc.). It will be evident that catalytic and/or highlyconserved residues are typically (but not necessarily) less preferredtargets for mutation.

Further, as noted above, a polymerase of the invention (e.g., a Φ29-typeDNA polymerase that includes E375, K512, L253, and/or A484 mutations)can be further modified to enhance the properties of the polymerase. Forexample, a polymerase comprising a combination of the above mutationscan be mutated at one or more additional sites to enhance a propertyalready possessed by the polymerase or to confer a new property notprovided by the existing mutations. Details correlating polymerasestructure with desirable functionalities that can be added topolymerases of the invention are provided herein. Also provide below arevarious approaches for modifying/mutating polymerases of the invention,determining kinetic parameters or other properties of the modifiedpolymerases, screening modified polymerases, and adding exogenousfeatures to the N- and/or C-terminal regions of the polymerases.

Structure-Based Design of Recombinant Polymerases

Structural data for a polymerase can be used to conveniently identifyamino acid residues as candidates for mutagenesis to create recombinantpolymerases, for example, having modified active site regions and/ormodified domain interfaces to increase phototolerance, reduce reactionrates, reduce branching, improve complex stability, reduce exonucleaseactivity, alter cofactor selectivity, increase stability, improve yield,or confer other desirable properties. For example, analysis of thethree-dimensional structure of a polymerase such as Φ29 can identifyresidues that are in the active polymerization site of the enzyme,residues that form part of the nucleotide analog binding pocket, and/oramino acids at an interface between domains.

The three-dimensional structures of a large number of DNA polymeraseshave been determined by x-ray crystallography and nuclear magneticresonance (NMR) spectroscopy, including the structures of polymeraseswith bound templates, nucleotides, and/or nucleotide analogs. Many suchstructures are freely available for download from the Protein Data Bank,at (www(dot)rcsb(dot)org/pdb. Structures, along with domain and homologyinformation, are also freely available for search and download from theNational Center for Biotechnology Information's Molecular ModelingDataBase, atwww(dot)ncbi(dot)nlm(dot)nih(dot)gov/Structure/MMDB/mmdb(dot)shtml. Thestructures of Φ29 polymerase, Φ29 polymerase complexed with terminalprotein, and Φ29 polymerase complexed with primer-template DNA in thepresence and absence of a nucleoside triphosphate are available; seeKamtekar et al. (2004) “Insights into strand displacement andprocessivity from the crystal structure of the protein-primed DNApolymerase of bacteriophage Φ29” Mol. Cell. 16(4): 609-618), Kamtekar etal. (2006) “The phi29 DNA polymerase:protein-primer structure suggests amodel for the initiation to elongation transition” EMBO J.25(6):1335-43, and Berman et al. (2007) “Structures of phi29 DNApolymerase complexed with substrate: The mechanism of translocation inB-family polymerases” EMBO J. 26:3494-3505, respectively. The structuresof additional polymerases or complexes can be modeled, for example,based on homology of the polymerases with polymerases whose structureshave already been determined. Alternatively, the structure of a givenpolymerase (e.g., a wild-type or modified polymerase), optionallycomplexed with a DNA (e.g., template and/or primer) and/or nucleotideanalog, or the like, can be determined.

Techniques for crystal structure determination are well known. See, forexample, McPherson (1999) Crystallization of Biological MacromoleculesCold Spring Harbor Laboratory; Bergfors (1999) Protein CrystallizationInternational University Line; Mullin (1993) CrystallizationButterwoth-Heinemann; Stout and Jensen (1989) X-ray structuredetermination: a practical guide, 2nd Edition Wiley Publishers, NewYork; Ladd and Palmer (1993) Structure determination by X-raycrystallography, 3rd Edition Plenum Press, NewYork; Blundell and Johnson(1976) Protein Crystallography Academic Press, New York; Glusker andTrueblood (1985) Crystal structure analysis: A primer, 2 nd Ed. OxfordUniversity Press, NewYork; International Tables for Crystallography,Vol. F. Crystallography of Biological Macromolecules; McPherson (2002)Introduction to Macromolecular Crystallography Wiley-Liss; McRee andDavid (1999) Practical Protein Crystallography, Second Edition AcademicPress; Drenth (1999) Principles of Protein X-Ray Crystallography(Springer Advanced Texts in Chemistry) Springer-Verlag; Fanchon andHendrickson (1991) Chapter 15 of Crystallographic Computing, Volume 5IUCr/Oxford University Press; Murthy (1996) Chapter 5 ofCrystallographic Methods and Protocols Humana Press; Dauter et al.(2000) “Novel approach to phasing proteins: derivatization by shortcryo-soaking with halides” Acta Cryst.D56:232-237; Dauter (2002) “Newapproaches to high-throughput phasing” Curr. Opin. Structural Biol.12:674-678; Chen et al. (1991) “Crystal structure of a bovineneurophysin-II dipeptide complex at 2.8 Å determined from thesingle-wavelength anomalous scattering signal of an incorporated iodineatom” Proc. Natl. Acad. Sci. USA, 88:4240-4244; and Gavira et al. (2002)“Ab initio crystallographic structure determination of insulin fromprotein to electron density without crystal handling” ActaCryst.D58:1147-1154.

In addition, a variety of programs to facilitate data collection, phasedetermination, model building and refinement, and the like are publiclyavailable. Examples include, but are not limited to, the HKL2000 package(Otwinowski and Minor (1997) “Processing of X-ray Diffraction DataCollected in Oscillation Mode” Methods in Enzymology 276:307-326), theCCP4 package (Collaborative Computational Project (1994) “The CCP4suite: programs for protein crystallography” Acta Crystallogr D50:760-763), SOLVE and RESOLVE (Terwilliger and Berendzen (1999) ActaCrystallogr D 55 (Pt 4):849-861), SHELXS and SHELXD (Schneider andSheldrick (2002) “Substructure solution with SHELXD” Acta Crystallogr DBiol Crystallogr 58:1772-1779), Refmac5 (Murshudov et al. (1997)“Refinement of Macromolecular Structures by the Maximum-LikelihoodMethod” Acta Crystallogr D 53:240-255), PRODRG (van Aalten et al. (1996)“PRODRG, a program for generating molecular topologies and uniquemolecular descriptors from coordinates of small molecules” J ComputAided Mol Des 10:255-262), and Coot (Elmsley et al. (2010) “Features andDevelopment of Coot” Acta Cryst D 66:486-501.

Techniques for structure determination by NMR spectroscopy are similarlywell described in the literature. See, e.g., Cavanagh et al. (1995)Protein NMR Spectroscopy: Principles and Practice, Academic Press;Levitt (2001) Spin Dynamics: Basics of Nuclear Magnetic Resonance, JohnWiley & Sons; Evans (1995) Biomolecular NMR Spectroscopy, OxfordUniversity Press; Wiithrich (1986) NMR of Proteins and Nucleic Acids(Baker Lecture Series), Kurt Wiley-Interscience; Neuhaus and Williamson(2000) The Nuclear Overhauser Effect in Structural and ConformationalAnalysis, 2nd Edition, Wiley-VCH; Macomber (1998) A CompleteIntroduction to Modern NMR Spectroscopy, Wiley-Interscience; Downing(2004) Protein NMR Techniques (Methods in Molecular Biology), 2ndedition, Humana Press; Clore and Gronenborn (1994) NMR of Proteins(Topics in Molecular and Structural Biology), CRC Press; Reid (1997)Protein NMR Techniques, Humana Press; Krishna and Berliner (2003)Protein NMR for the Millenium (Biological Magnetic Resonance), KluwerAcademic Publishers; Kiihne and De Groot (2001) Perspectives on SolidState NMR in Biology (Focus on Structural Biology, 1), Kluwer AcademicPublishers; Jones et al. (1993) Spectroscopic Methods and Analyses: NMR,Mass Spectrometry, and Related Techniques (Methods in Molecular Biology,Vol. 17), Humana Press; Goto and Kay (2000) Curr. Opin. Struct. Biol.10:585; Gardner (1998) Annu. Rev. Biophys. Biomol. Struct. 27:357;Wiithrich (2003) Angew. Chem. Int. Ed. 42:3340; Bax (1994) Curr. Opin.Struct. Biol. 4:738; Pervushin et al. (1997) Proc. Natl. Acad. Sci.U.S.A. 94:12366; Fiaux et al. (2002) Nature 418:207; Fernandez and Wider(2003) Curr. Opin. Struct. Biol. 13:570; Ellman et al. (1992) J. Am.Chem. Soc. 114:7959; Wider (2000) BioTechniques 29:1278-1294; Pellecchiaet al. (2002) Nature Rev. Drug Discov. (2002) 1:211-219; Arora and Tamm(2001) Curr. Opin. Struct. Biol. 11:540-547; Flaux et al. (2002) Nature418:207-211; Pellecchia et al. (2001) J. Am. Chem. Soc. 123:4633-4634;and Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA 94:12366-12371.

The structure of a polymerase or of a polymerase bound to a DNA or witha given nucleotide analog incorporated into the active site can, asnoted, be directly determined, e.g., by x-ray crystallography or NMRspectroscopy, or the structure can be modeled based on the structure ofthe polymerase and/or a structure of a polymerase with a naturalnucleotide bound. The active site or other relevant domain of thepolymerase can be identified, for example, by homology with otherpolymerases, examination of polymerase-template or polymerase-nucleotideco-complexes, biochemical analysis of mutant polymerases, and/or thelike. The position of a nucleotide analog (as opposed to an availablenucleotide structure) in the active site can be modeled, for example, byprojecting the location of non-natural features of the analog (e.g.,additional phosphate or phosphonate groups in the phosphorus containingchain linked to the nucleotide, e.g., tetra, penta or hexa phosphategroups, detectable labeling groups, e.g., fluorescent dyes, or the like)based on the previously determined location of another nucleotide ornucleotide analog in the active site.

Such modeling of the nucleotide analog or template (or both) in theactive site can involve simple visual inspection of a model of thepolymerase, for example, using molecular graphics software such as thePyMOL viewer (open source, freely available on the World Wide Web atwww(dot)pymol(dot)org), Insight II, or Discovery Studio 2.1(commercially available from Accelrys at (www (dot) accelrys (dot)com/products/discovery-studio). Alternatively, modeling of the activesite complex of the polymerase or a putative mutant polymerase, forexample, can involve computer-assisted docking, molecular dynamics, freeenergy minimization, and/or like calculations. Such modeling techniqueshave been well described in the literature; see, e.g., Babine andAbdel-Meguid (eds.) (2004) Protein Crystallography in Drug Design,Wiley-VCH, Weinheim; Lyne (2002) “Structure-based virtual screening: Anoverview” Drug Discov. Today 7:1047-1055; Molecular Modeling forBeginners, at (www (dot) usm (dot) maine (dot) edu/˜rhodes/SPVTut/index(dot) html; and Methods for Protein Simulations and Drug Design at (www(dot) dddc (dot) ac (dot) cn/embo04; and references therein. Software tofacilitate such modeling is widely available, for example, the CHARMmsimulation package, available academically from Harvard University orcommercially from Accelrys (at www (dot) accelrys (dot) com), theDiscover simulation package (included in Insight II, supra), and Dynama(available at (www(dot) cs (dot) gsu (dot) edu/˜cscrwh/progs/progs (dot)html). See also an extensive list of modeling software at (www (dot)netsci (dot) org/Resources/Software/Modeling/MMMD/top (dot) html.

Visual inspection and/or computational analysis of a polymerase model,including optional comparison of models of the polymerase in differentstates, can identify relevant features of the polymerase, including, forexample, residues that can be mutated to increase phototolerance orpolymerase speed, as detailed above.

In another example, residues from domains that are in close proximity toone another are mutated to alter inter-domain interactions. In Φ29, Q183in the exonuclease domain can contact the back of the fingers domain(e.g., Q183 is close to 1378, particularly when the fingers are open).Mutating this residue can thus alter the equilibrium between the openand closed conformations of the polymerase. A Q183F substitution, forexample, significantly increases mean readlength, although it alsoreduces accuracy somewhat. This substitution can therefore be ofinterest in polymerases for applications where readlength is of greaterpriority than accuracy, e.g., for scaffolding genome assembly. Othersubstitutions at this position include, e.g., Q183W and Q183T (whichalso increase readlength), as well as Q183H.

As described in U.S. patent application publication 2012-0034602,substitutions at position L253 of Φ29 can affect cofactor selectivity.Introducing an A437G substitution into the polymerase can increasepolymerase speed and can also increase the range of useful substitutionsat position L253. For example, a combination of L253H and A437Gsubstitutions can reduce pulse width and pausing, increase readlength,and enhance Mg⁺⁺ tolerance. As shown in FIG. 8, examination of a crystalstructure of a recombinant Φ29 polymerase including D12A, D66A, Y224K,E239G, L253H, E375Y, A437G, A484E, D510K, and K512Y substitutionsreveals that the histidine at position 253 forms a hydrogen bond withthe backbone carbonyl of residue 437. Formation of the hydrogen bond isenabled by the A437G substitution.

Substitution of V250 can also increase the range of functionalsubstitutions at position L253. For example, replacement of valine atposition 250 with a smaller residue, e.g., alanine, can accommodate alarger side chain at position L253. Exemplary combinations include,e.g., V250A with L253H or L253F. A V250A substitution can also increasereadlength.

Amino acid sequence data, e.g., for members of a family of polymerases,can be used in conjunction with structural data to identify particularresidues as candidates for mutagenesis. As one example, residues thatdiffer between family members and that are close to the active site canbe mutated. For example, as shown in FIG. 1, wild-type Φ29 has analanine at position 256 while wild-type M2Y has a serine at thecorresponding position (position 253 of M2Y, SEQ ID NO:2). Introducingan S253A substitution into M2Y, where positions are numbered withrespect to SEQ ID NO:2, can increase readlength and decrease pulsewidth, improving performance in single molecule sequencing assays. AnA256S substitution can be introduced into Φ29, where positions arenumbered with respect to SEQ ID NO:1, e.g., to increase pulse width. Asanother example, wild-type Φ29 has a tyrosine at position 224 whilewild-type M2Y has a lysine at the corresponding position (position 221of M2Y, SEQ ID NO:2). A Y224K substitution can be introduced into Φ29,where positions are numbered with respect to SEQ ID NO:1, or a K221Ysubstitution can be introduced into M2Y, where positions are numberedwith respect to SEQ ID NO:2.

Combining Mutations

As noted repeatedly, the various mutations described herein can becombined in recombinant polymerases of the invention. Combination ofmutations can be random, or more desirably, guided by the properties ofthe particular mutations and the characteristics desired for theresulting polymerase. Additional mutations can also be introduced into apolymerase to compensate for deleterious effects of otherwise desirablemutations.

A large number of exemplary mutations and the properties they confer aredescribed herein, and it will be evident that these mutations can befavorably combined in many different combinations. Exemplarycombinations are also provided herein, e.g., in Tables 3 and 4 and FIG.7, and an example of strategies by which additional favorablecombinations are readily derived follows. For the sake of simplicity, afew exemplary combinations using only a few exemplary mutations arediscussed, but it will be evident that any of the mutations describedherein can be employed in such strategies to produce polymerases withdesirable properties.

For example, where a recombinant polymerase is desired to incorporatephosphate-labeled phosphate analogs in a Mg⁺⁺-containing single moleculesequencing reaction, one or more substitutions that enhance analogbinding (e.g., E375Y, K512Y, and/or A484E) and one or more substitutionsthat alter metal cofactor usage (e.g., L253A, L253H, or L253S) can beincorporated. One or more substitutions that increase phototolerance(e.g., K131E, K131Q, and/or K135Q) can be included. Exemplarycombinations thus include K131E, L253A and A484E; K131E, L253A, E375Y,and K512Y; K131E, L253A, E375Y, A484E, and K512Y; K135Q, L253A andA484E; K135Q, L253A, E375Y, and K512Y; and K135Q, L253A, E375Y, A484E,and K512Y. Polymerase speed can be enhanced by inclusion ofsubstitutions such as A437G, E508K, V141K, L142K, D510K, and/or V250I,providing combinations such as A437G, L253A, and A484E; A437G, E375Y,and K512Y; K131E, L253A, A484E, and D510K; K135Q, L253A, A484E, andD510K; K131E, Y148I, L253A, and A484E; K135Q, Y148I, L253A, and A484E;K131E, Y148I, L253A, E375Y, A484E, and K512Y; and K135Q, Y148I, L253A,E375Y, A484E, and K512Y. Stability and/or yield can be increased byinclusion of substitutions such as E239G, V250I, and/or Y224K, producingcombinations such as K131E, E239G, L253A, A484E, and D510K; K135Q,E239G, L253A, A484E, and D510K; K131E, E239G, L253A, E375Y, A484E,D510K, and K512Y; K135Q, E239G, L253A, E375Y, A484E, D510K, and K512Y;K131E, Y224K, E239G, L253A, E375Y, A484E, D510K, and K512Y; and K135Q,Y224K, E239G, L253A, E375Y, A484E, D510K, and K512Y. Accuracy can beenhanced by inclusion of substitutions such as E515Q, D235E, and/orY148I, providing combinations such as K131E, Y148I, Y224K, E239G, V250I,L253A, E375Y, A484E, D510K, and K512Y; K131E, Y148I, Y224K, E239G,V250I, L253H, E375Y, A437G, A484E, and K512Y; K135Q, Y148I, Y224K,E239G, V250I, L253H, E375Y, A437G, A484E, and K512Y; K131E, Y148I,Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y, andE515Q; K135Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,D510K, K512Y, and E515Q; K135Q, Y148I, Y224K, E239G, V250I, L253A,E375Y, A484E, D510K, and K512Y; K131E, Y148I, Y224K, E239G, V250I,L253A, E375Y, A484E, D510K, K512Y, and E515Q; and K135Q, Y148I, Y224K,E239G, V250I, L253A, E375Y, A484E, D510K, K512Y, and E515Q.

Additional exemplary combinations of substitutions that can be presentin a polymerase of the invention include, but are not limited to: E239G,L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; E239G, V250I,L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; E239G, L253A,E375Y, A437N, A484E, D510K, K512Y, and E515Q; E239G, V250I, L253A,E375Y, A437N, A484E, D510K, K512Y, and E515Q; E239G, V250A, L253H,E375Y, A437G, A484E, D510K, K512Y, and E515Q; E239G, V250A, L253H,E375Y, A437N, A484E, D510K, K512Y, and E515Q; Y224K, E239G, L253A,E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y224K, E239G, V250I,L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; Y224K, E239G,L253A, E375Y, A437N, A484E, D510K, K512Y, and E515Q; Y224K, E239G,V250I, L253A, E375Y, A437N, A484E, D510K, K512Y, and E515Q; Y224K,E239G, V250A, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q;Y224K, E239G, V250A, L253H, E375Y, A437N, A484E, D510K, K512Y, andE515Q; K131E, E239G, L253A, E375Y, A437G, A484E, D510K, K512Y, andE515Q; K131E, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y,and E515Q; K131E, E239G, L253A, E375Y, A437N, A484E, D510K, K512Y, andE515Q; K131E, E239G, V250I, L253A, E375Y, A437N, A484E, D510K, K512Y,and E515Q; K131E, E239G, V250A, L253H, E375Y, A437G, A484E, D510K,K512Y, and E515Q; K131E, E239G, V250A, L253H, E375Y, A437N, A484E,D510K, K512Y, and E515Q; K131E, Y224K, E239G, L253A, E375Y, A437G,A484E, D510K, K512Y, and E515Q; K131E, Y224K, E239G, V250I, L253A,E375Y, A437G, A484E, D510K, K512Y, and E515Q; K131E, Y224K, E239G,L253A, E375Y, A437N, A484E, D510K, K512Y, and E515Q; K131E, Y224K,E239G, V250I, L253A, E375Y, A437N, A484E, D510K, K512Y, and E515Q;K131E, Y224K, E239G, V250A, L253H, E375Y, A437G, A484E, D510K, K512Y,and E515Q; and K131E, Y224K, E239G, V250A, L253H, E375Y, A437N, A484E,D510K, K512Y, and E515Q.

Many other such recombinant polymerases, including these mutationsand/or those described elsewhere herein, will be readily apparent andare features of the invention.

Mutating Polymerases

Various types of mutagenesis are optionally used in the presentinvention, e.g., to modify polymerases to produce variants, e.g., inaccordance with polymerase models and model predictions as discussedabove, or using random or semi-random mutational approaches. In general,any available mutagenesis procedure can be used for making polymerasemutants. Such mutagenesis procedures optionally include selection ofmutant nucleic acids and polypeptides for one or more activity ofinterest (e.g., increased phototolerance, reduced reaction rates,decreased exonuclease activity, increased complex stability, decreasedbranching fraction, altered metal cofactor selectivity, improvedprocessivity, increased thermostability, increased yield, increasedaccuracy, and/or improved k_(off), K_(m), V_(max), k_(cat) etc., e.g.,for a given nucleotide analog). Procedures that can be used include, butare not limited to: site-directed point mutagenesis, random pointmutagenesis, in vitro or in vivo homologous recombination (DNA shufflingand combinatorial overlap PCR), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA, point mismatch repair, mutagenesis using repair-deficienthost strains, restriction-selection and restriction-purification,deletion mutagenesis, mutagenesis by total gene synthesis, degeneratePCR, double-strand break repair, and many others known to persons ofskill. The starting polymerase for mutation can be any of those notedherein, including available polymerase mutants such as those identifiede.g., in WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUEINCORPORATION by Hanzel et al.; WO 2008/051530 POLYMERASE ENZYMES ANDREAGENTS FOR ENHANCED NUCLEIC ACID SEQUENCING; U.S. patent applicationpublication 2010-0075332 ENGINEERING POLYMERASES AND REACTION CONDITIONSFOR MODIFIED INCORPORATION PROPERTIES by Pranav Patel et al.; U.S.patent application publication 2010-0093555 ENZYMES RESISTANT TOPHOTODAMAGE by Keith Bjornson et al.; U.S. patent applicationpublication 2010-0112645 GENERATION OF MODIFIED POLYMERASES FOR IMPROVEDACCURACY 1N SINGLE MOLECULE SEQUENCING by Sonya Clark et al.; U.S.patent application publication 2011-0189659 GENERATION OF MODIFIEDPOLYMERASES FOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCING by SonyaClark et al.; U.S. patent application publication 2012-0034602RECOMBINANT POLYMERASES FOR IMPROVED SINGLE MOLECULE SEQUENCING; Hanzelet al. WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES; and Hanzel etal. 2007/075873 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OFSURFACE ATTACHED PROTEINS.

Optionally, mutagenesis can be guided by known information from anaturally occurring polymerase molecule, or of a known altered ormutated polymerase (e.g., using an existing mutant polymerase as notedin the preceding references), e.g., sequence, sequence comparisons,physical properties, crystal structure and/or the like as discussedabove. However, in another class of embodiments, modification can beessentially random (e.g., as in classical or “family” DNA shuffling,see, e.g., Crameri et al. (1998) “DNA shuffling of a family of genesfrom diverse species accelerates directed evolution” Nature391:288-291).

Additional information on mutation formats is found in: Sambrook et al.,Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2012) (“Ausubel”))and PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (“Innis”). The followingpublications and references cited within provide additional detail onmutation formats: Arnold, Protein engineering for unusual environments,Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., MutantTrp repressors with new DNA-binding specificities, Science 242:240-245(1988); Bordo and Argos (1991) Suggestions for “Safe” ResidueSubstitutions in Site-directed Mutagenesis 217:721-729; Botstein &Shortle, Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter et al., Improved oligonucleotidesite-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13:4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237:1-7(1986); Carter, Improved oligonucleotide-directed mutagenesis using M13vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff,Use of oligonucleotides to generate large deletions, Nucl. Acids Res.14: 5115 (1986); Fritz et al., Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro, Nucl. Acids Res. 16: 6987-6999 (1988); Grundstrom et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Hayes (2002) CombiningComputational and Experimental Screening for rapid Optimization ofProtein Properties PNAS 99(25) 15926-15931; Kunkel, The efficiency ofoligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D.M.J. eds., Springer Verlag, Berlin))(1987); Kunkel, Rapid and efficient site-specific mutagenesis withoutphenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492 (1985);Kunkel et al., Rapid and efficient site-specific mutagenesis withoutphenotypic selection, Methods in Enzymol. 154, 367-382 (1987); Kramer etal., The gapped duplex DNA approach to oligonucleotide-directed mutationconstruction, Nucl. Acids Res. 12: 9441-9456 (1984); Kramer & FritzOligonucleotide-directed construction of mutations via gapped duplexDNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al., PointMismatch Repair, Cell 38:879-887 (1984); Kramer et al., Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations, Nucl. Acids Res. 16:7207 (1988); Ling et al., Approaches to DNA mutagenesis: an overview,Anal Biochem. 254(2): 157-178 (1997); Lorimer and Pastan Nucleic AcidsRes. 23, 3067-8 (1995); Mandecki, Oligonucleotide-directed double-strandbreak repair in plasmids of Escherichia coli: a method for site-specificmutagenesis, Proc. Natl. Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye &Eckstein, Inhibition of restriction endonuclease Nci I cleavage byphosphorothioate groups and its application to oligonucleotide-directedmutagenesis, Nucl. Acids Res. 14: 9679-9698 (1986); Nambiar et al.,Total synthesis and cloning of a gene coding for the ribonuclease Sprotein, Science 223: 1299-1301 (1984); Sakamar and Khorana, Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl. AcidsRes. 14: 6361-6372 (1988); Sayers et al., Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Sieber, et al., Nature Biotechnology, 19:456-460(2001); Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985);Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154:329-350 (1987); Stemmer, Nature 370, 389-91 (1994); Taylor et al., Theuse of phosphorothioate-modified DNA in restriction enzyme reactions toprepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor etal., The rapid generation of oligonucleotide-directed mutations at highfrequency using phosphorothioate-modified DNA, Nucl. Acids Res. 13:8765-8787 (1985); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Wells et al., Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites,Gene 34:315-323 (1985); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Clackson et al. (1991) “Making antibodyfragments using phage display libraries” Nature 352:624-628; Gibbs etal. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): a methodfor enhancing the frequency of recombination with family shuffling” Gene271:13-20; and Hiraga and Arnold (2003) “General method forsequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296. Additional details on many of the above methods can befound in Methods in Enzymology Volume 154, which also describes usefulcontrols for trouble-shooting problems with various mutagenesis methods.

Determining Kinetic Parameters

The polymerases of the invention can be screened or otherwise tested todetermine whether the polymerase displays a modified activity for orwith a nucleotide analog or template as compared to a parental DNApolymerase (e.g., a corresponding wild-type or available mutantpolymerase from which the recombinant polymerase of the invention wasderived). For example, branching fraction, a reaction rate constant,k_(off), k_(cat), K_(m), V_(max), k_(cat)/K_(m), V_(max)/K_(m), k_(pol),and/or K_(d) of the recombinant DNA polymerase for the nucleotide (oranalog) or template nucleic acid can be determined. The specificityconstant k_(cat)/K_(m) is also a useful measure, e.g., for assessingbranch rate. k_(cat)/K_(m) is a measure of substrate binding that leadsto product formation (and, thus, includes terms defining binding K_(d)and inversely predicts branching fraction formation).

As is well-known in the art, for enzymes obeying simple Michaelis-Mentenkinetics, kinetic parameters are readily derived from rates of catalysismeasured at different substrate concentrations. The Michaelis-Mentenequation, V=V_(max)[S]([S]+K_(m))⁻¹, relates the concentration of freesubstrate ([S], approximated by the total substrate concentration), themaximal rate (V_(max), attained when the enzyme is saturated withsubstrate), and the Michaelis constant (K_(m), equal to the substrateconcentration at which the reaction rate is half of its maximal value),to the reaction rate (V).

For many enzymes, K_(m) is equal to the dissociation constant of theenzyme-substrate complex and is thus a measure of the strength of theenzyme-substrate complex. For such an enzyme, in a comparison of K_(m)s,a lower K_(m) represents a complex with stronger binding, while a higherKm represents a complex with weaker binding. The ratio k_(cat)/K_(m),sometimes called the specificity constant, can be thought of as thesecond order rate constant times the probability of that substrate beingconverted to product once bound. The larger the specificity constant,the more efficient the enzyme is in binding the substrate and convertingit to product. The specificity constant is inversely proportional to thebranching rate, as branching rate is the rate at which the enzyme bindssubstrate (e.g., nucleotide) but does not convert it to product (e.g., aDNA polymer).

k_(cat) (also called the turnover number of the enzyme) can bedetermined if the total enzyme concentration ([E_(T)], i.e., theconcentration of active sites) is known, since V_(max)=k_(cat)[E_(T)].For situations in which the total enzyme concentration is difficult tomeasure, the ratio V_(max)/K_(m) is often used instead as a measure ofefficiency. K_(m) and V_(max) can be determined, for example, from aLineweaver-Burk plot of 1/V against 1/[S], where the y interceptrepresents 1/V_(max), the x intercept −1/K_(m), and the slopeK_(m)/V_(max), or from an Eadie-Hofstee plot of V against V/[S], wherethe y intercept represents V_(max), the x intercept V_(max)/K_(m), andthe slope −K_(m). Software packages such as KinetAsyst™ or Enzfit(Biosoft, Cambridge, UK) can facilitate the determination of kineticparameters from catalytic rate data.

For enzymes such as polymerases that have multiple substrates, varyingthe concentration of only one substrate while holding the others insuitable excess (e.g., effectively constant) concentration typicallyyields normal Michaelis-Menten kinetics.

Details regarding k_(off) determination are described, e.g., in U.S.patent application publication 2012-0034602. In general, thedissociation rate can be measured in any manner that detects thepolymerase/DNA complex over time. This includes stopped-flowspectroscopy, or even simply taking aliquots over time and testing forpolymerase activity on the template of interest. Free polymerase iscaptured with a polymerase trap after dissociation, e.g., by incubationin the presence of heparin or an excess of competitor DNA (e.g.,non-specific salmon sperm DNA, or the like).

In one embodiment, using pre-steady-state kinetics, the nucleotideconcentration dependence of the rate constant k_(obs) (the observedfirst-order rate constant for dNTP incorporation) provides an estimateof the K_(m) for a ground state binding and the maximum rate ofpolymerization (k_(pol)). The k_(obs) is measured using a burst assay.The results of the assay are fitted with the Burst equation;Product=A[1-exp(−k_(obs)*t)]+k_(ss)*t where A represents amplitude anestimate of the concentration of the enzyme active sites, k_(ss) is theobserved steady-state rate constant and t is the reaction incubationtime. The K_(m) for dNTP binding to the polymerase-DNA complex and thek_(pol) are calculated by fitting the dNTP concentration dependentchange in the k_(obs) using the equationk_(obs)=(k_(pol)*[S])*(K_(m)+[S])⁻¹ where [S] is the substrateconcentration. Results are optionally obtained from a rapid-quenchexperiment (also called a quench-flow measurement), for example, basedon the methods described in Johnson (1986) “Rapid kinetic analysis ofmechanochemical adenosinetriphosphatases” Methods Enzymol. 134:677-705,Patel et al. (1991) “Pre-steady-state kinetic analysis of processive DNAreplication including complete characterization of anexonuclease-deficient mutant” Biochemistry 30(2):511-25, and Tsai andJohnson (2006) “A new paradigm for DNA polymerase specificity”Biochemistry 45(32):9675-87.

Parameters such as rate of binding of a nucleotide analog or template bythe recombinant polymerase, rate of product release by the recombinantpolymerase, or branching rate of the recombinant polymerase can also bedetermined, and optionally compared to that of a parental polymerase(e.g., a corresponding wild-type polymerase).

For a more thorough discussion of enzyme kinetics, see, e.g., Berg,Tymoczko, and Stryer (2002) Biochemistry, Fifth Edition, W.H. Freeman;Creighton (1984) Proteins: Structures and Molecular Principles, W.H.Freeman; and Fersht (1985) Enzyme Structure and Mechanism, SecondEdition, W.H. Freeman. In one aspect, the improved activity of theenzymes of the invention is compared with a given parental polymerase.For example, in the case of enzymes derived from a Φ29 parental enzyme,where the improvement being sought is an increase in stability of theclosed complex, an improved enzyme of the invention would have a lowerk_(off) than the parental enzyme, e.g., wild type Φ29. Such comparisonsare made under equivalent reaction conditions, e.g., equalconcentrations of the parental and modified polymerase, equal substrateconcentrations, equivalent solution conditions (pH, salt concentration,presence of divalent cations, etc.), temperature, and the like. In oneaspect, the improved activity of the enzymes of the invention ismeasured with reference to a model analog or analog set and comparedwith a given parental enzyme. Optionally, the improved activity of theenzymes of the invention is measured under specified reactionconditions. While the foregoing may be used as a characterization tool,it in no way is intended as a specifically limiting reaction of theinvention.

Optionally, the polymerase exhibits a K_(m) for a phosphate-labelednucleotide analog that is less than a K_(m) observed for a wild-typepolymerase for the analog to facilitate applications in which thepolymerase incorporates the analog, e.g., during SMS. For example, themodified recombinant polymerase can exhibit a K_(m) for thephosphate-labeled nucleotide analog that is less than 75%, less than50%, or less than 25% than that of wild-type or parental polymerase suchas a wild type Φ29. In one specific class of examples, the polymerasesof the invention have a K_(m) of about 10 μM or less for a non-naturalnucleotide analog such as a phosphate labeled analog.

Screening Polymerases

Screening or other protocols can be used to determine whether apolymerase displays a modified activity, e.g., for a nucleotide analog,as compared to a parental DNA polymerase. For example, branchingfraction, rate constant, k_(off), k_(cat), K_(m), V_(max), ork_(cat)/K_(m) of the recombinant DNA polymerase for the template ornucleotide or analog can be determined as discussed above. As anotherexample, activity can be assayed indirectly. Assays for properties suchas protein yield, thermostability, and the like are described, e.g., inU.S. patent application publication 2012-0034602. Performance of arecombinant polymerase in a sequencing reaction, e.g., a single moleculesequencing reaction, can be examined to assay properties such as speed,pulse width, interpulse distance, accuracy, readlength, etc. asdescribed herein. Phototolerance can be assessed by monitoringpolymerase performance (e.g., in a single molecule sequencing reaction)during or after exposure of the polymerase to light, e.g., excitationlight of a specified wavelength at a given intensity for a given time,e.g., as compared to a wild-type or other parental polymerase.

In one desirable aspect, a library of recombinant DNA polymerases can bemade and screened for these properties. For example, a plurality ofmembers of the library can be made to include one or more mutation thatincreases phototolerance, alters (e.g., decreases) reaction rateconstants, improves closed complex stability, decreases branchingfraction, alters cofactor selectivity, or increases yield,thermostability, accuracy, speed, or readlength and/or randomlygenerated mutations (e.g., where different members include differentmutations or different combinations of mutations), and the library canthen be screened for the properties of interest (e.g., increasedphototolerance, decreased rate constant, decreased branching fraction,increased closed complex stability, etc.). In general, the library canbe screened to identify at least one member comprising a modifiedactivity of interest.

Libraries of polymerases can be either physical or logical in nature.Moreover, any of a wide variety of library formats can be used. Forexample, polymerases can be fixed to solid surfaces in arrays ofproteins. Similarly, liquid phase arrays of polymerases (e.g., inmicrowell plates) can be constructed for convenient high-throughputfluid manipulations of solutions comprising polymerases. Liquid,emulsion, or gel-phase libraries of cells that express recombinantpolymerases can also be constructed, e.g., in microwell plates, or onagar plates. Phage display libraries of polymerases or polymerasedomains (e.g., including the active site region or interdomain stabilityregions) can be produced. Likewise, yeast display libraries can be used.Instructions in making and using libraries can be found, e.g., inSambrook, Ausubel and Berger, referenced herein.

For the generation of libraries involving fluid transfer to or frommicrotiter plates, a fluid handling station is optionally used. Several“off the shelf” fluid handling stations for performing such transfersare commercially available, including e.g., the Zymate systems fromCaliper Life Sciences (Hopkinton, Mass.) and other stations whichutilize automatic pipettors, e.g., in conjunction with the robotics forplate movement (e.g., the ORCA® robot, which is used in a variety oflaboratory systems available, e.g., from Beckman Coulter, Inc.(Fullerton, Calif.).

In an alternate embodiment, fluid handling is performed in microchips,e.g., involving transfer of materials from microwell plates or otherwells through microchannels on the chips to destination sites(microchannel regions, wells, chambers or the like). Commerciallyavailable microfluidic systems include those fromHewlett-Packard/Agilent Technologies (e.g., the HP2100 bioanalyzer) andthe Caliper High Throughput Screening System. The Caliper HighThroughput Screening System provides one example interface betweenstandard microwell library formats and Labchip technologies. RainDanceTechnologies' nanodroplet platform provides another method for handlinglarge numbers of spatially separated reactions. Furthermore, the patentand technical literature includes many examples of microfluidic systemswhich can interface directly with microwell plates for fluid handling.

Tags and Other Optional Polymerase Features

The recombinant DNA polymerase optionally includes additional featuresexogenous or heterologous to the polymerase. For example, therecombinant polymerase optionally includes one or more tags, e.g.,purification, substrate binding, or other tags, such as a polyhistidinetag, a His10 tag, a His6 tag, an alanine tag, an Ala10 tag, an A1a16tag, a biotin tag, a biotin ligase recognition sequence or other biotinattachment site (e.g., a BiTag or a Btag or variant thereof, e.g.,BtagV1-11), a GST tag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D)tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising theamino acids glycine and serine, a tag or linker comprising the aminoacids glycine, serine, alanine and histidine, a tag or linker comprisingthe amino acids glycine, arginine, lysine, glutamine and proline, aplurality of polyhistidine tags, a plurality of His10 tags, a pluralityof His6 tags, a plurality of alanine tags, a plurality of Ala10 tags, aplurality of A1a16 tags, a plurality of biotin tags, a plurality of GSTtags, a plurality of BiTags, a plurality of S Tags, a plurality ofSNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, aplurality of lysine tags, a plurality of NanoTags, a plurality of Cmyctags, a plurality of tags or linkers comprising the amino acids glycineand serine, a plurality of tags or linkers comprising the amino acidsglycine, serine, alanine and histidine, a plurality of tags or linkerscomprising the amino acids glycine, arginine, lysine, glutamine andproline, biotin, avidin, an antibody or antibody domain, antibodyfragment, antigen, receptor, receptor domain, receptor fragment, maltosebinding protein, ligand, one or more protease site (e.g., Factor Xa,enterokinase, or thrombin site), a dye, an acceptor, a quencher, a DNAbinding domain (e.g., a helix-hairpin-helix domain from topoisomeraseV), or combination thereof. See, e.g., U.S. patent applicationpublication 2012-0034602 for sequences of a number of suitable tags andlinkers, including Btag V1-11. The one or more exogenous or heterologousfeatures can find use not only for purification purposes, immobilizationof the polymerase to a substrate, and the like, but can also be usefulfor altering one or more properties of the polymerase.

The one or more exogenous or heterologous features can be includedinternal to the polymerase, at the N-terminal region of the polymerase,at the C-terminal region of the polymerase, or at a combination thereof(e.g., at both the N-terminal and C-terminal regions of the polymerase).Where the polymerase includes an exogenous or heterologous feature atboth the N-terminal and C-terminal regions, the exogenous orheterologous features can be the same (e.g., a polyhistidine tag, e.g.,a His10 tag, at both the N- and C-terminal regions) or different (e.g.,a biotin ligase recognition sequence at the N-terminal region and apolyhistidine tag, e.g., His10 tag, at the C-terminal region).Optionally, a terminal region (e.g., the N- or C-terminal region) of apolymerase of the invention can comprise two or more exogenous orheterologous features which can be the same or different (e.g., a biotinligase recognition sequence and a polyhistidine tag at the N-terminalregion, a biotin ligase recognition sequence, a polyhistidine tag, and aFactor Xa recognition site at the N-terminal region, and the like). As afew examples, the polymerase can include a polyhistidine tag at theC-terminal region, a biotin ligase recognition sequence at theN-terminal region and a polyhistidine tag at the C-terminal region, abiotin ligase recognition sequence and a polyhistidine tag at theN-terminal region, a biotin ligase recognition sequence and apolyhistidine tag at the N-terminal region and a polyhistidine tag atthe C-terminal region, or a polyhistidine tag and a biotin ligaserecognition sequence at the C-terminal region.

For convenience, an exogenous or heterologous feature will often beexpressed as a fusion domain of the overall polymerase protein, e.g., asa conventional in-frame fusion of a polypeptide sequence with the activepolymerase enzyme (e.g., a polyhistidine tag fused in frame to an activepolymerase enzyme sequence). However, features such as tags can be addedchemically to the polymerase, e.g., by using an available amino acidresidue of the enzyme or by incorporating an amino acid into the proteinthat provides a suitable attachment site for the coupling domain.Suitable residues of the enzyme can include, e.g., histidine, cysteine,or serine residues (providing for N, S, or O linked coupling reactions).Optionally, one or more cysteines present in the parental polymerase(e.g., up to all of the cysteines present on the polymerase's surface)can be replaced with a different amino acid; either a single reactivesurface cysteine can be left unsubstituted or a single reactive surfacecysteine can be introduced in place of another residue, for convenientaddition of a feature, e.g., for surface immobilization through thiollabeling (e.g., addition of maleimide biotin, or maleimide and an alkynefor click labeling). Unnatural amino acids that comprise unique reactivesites can also be added to the enzyme, e.g., by expressing the enzyme ina system that comprises an orthogonal tRNA and an orthogonal synthetasethat loads the unnatural amino acid in response to a selector codon.

The exogenous or heterologous features can find use, e.g., in thecontext of binding a polymerase in an active form to a surface, e.g., toorient and/or protect the polymerase active site when the polymerase isbound to a surface. In general, surface binding elements andpurification tags that can be added to the polymerase (e.g.,recombinantly or chemically) include, e.g., biotin attachment sites(e.g., biotin ligase recognition sequences such as Btags or BiTag),polyhistidine tags, His6 tags, His10 tags, biotin, avidin, GSTsequences, modified GST sequences, e.g., that are less likely to formdimers, S tags, SNAP-tags, antibodies or antibody domains, antibodyfragments, antigens, receptors, receptor domains, receptor fragments,ligands, and combinations thereof.

One aspect of the invention includes DNA polymerases that can be coupledto a surface without substantial loss of activity (e.g., in an activeform). DNA polymerases can be coupled to the surface through a singlesurface coupling domain or through multiple surface coupling domainswhich act in concert to increase binding affinity of the polymerase forthe surface and to orient the polymerase relative to the surface. Forexample, the active site can be oriented distal to the surface, therebymaking it accessible to a polymerase substrate (template, nucleotides,etc.). This orientation also tends to reduce surface denaturationeffects in the region of the active site. In a related aspect, activityof the enzyme can be protected by making the coupling domains large,thereby serving to further insulate the active site from surface bindingeffects. Further details regarding the immobilization of a polymerase toa surface (e.g., the surface of a zero mode waveguide) in an active formare found in WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzelet al., and WO 2007/075873 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZEACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al. Further detailson attaching tags is available in the art. See, e.g., U.S. Pat. Nos.5,723,584 and 5,874,239 and U.S. patent application publication2011/0306096 for additional information on attaching biotinylationpeptides to recombinant proteins.

The polymerase immobilized on a surface in an active form can be coupledto the surface through one or a plurality of artificial or recombinantsurface coupling domains as discussed above, and typically displays ak_(cat)/K_(m) (or V_(max)/K_(m)) that is at least about 1%, at leastabout 10%, at least about 25%, at least about 50%, or at least about 75%as high as a corresponding active polymerase in solution.

Exonuclease-Deficient Recombinant Polymerases

Many native DNA polymerases have a proof-reading exonuclease functionwhich can yield substantial data analysis problems in processes thatutilize real time observation of incorporation events as a method ofidentifying sequence information, e.g., single molecule sequencingapplications. Even where exonuclease activity does not introduce suchproblems in single molecule sequencing, reduction of exonucleaseactivity can be desirable since it can increase accuracy (in some casesat the expense of readlength).

Accordingly, recombinant polymerases of the invention optionally includeone or more mutations (e.g., substitutions, insertions, and/ordeletions) relative to the parental polymerase that reduce or eliminateendogenous exonuclease activity. For example, relative to the wild-typeΦ29 DNA polymerase of SEQ ID NO:1, one or more of positions N62, D12,E14, T15, H61, D66, D169, K143, Y148, and H149 is optionally mutated toreduce exonuclease activity. Exemplary mutations that can reduceexonuclease activity include, e.g., N62D, N62H, D12A, T15I, E14I, E14A,D66A, K143D, D145A and D169A substitutions, as well as addition of anexogenous feature at the C-terminus (e.g., a polyhistidine tag).Additional exemplary substitutions in the exonuclease domain includeN62S, D12N, D12R, D12M, E14Q, H61K, H61D, H61A, D66R, D66N, D66Q, D66K,D66M, D169N, K143R, Y148I, Y148K, Y148A, Y148C, Y148D, Y148E, Y148F,Y148G, Y148H, Y148L, Y148M, Y148N, Y148P, Y148Q, Y148R, Y148S, Y148T,Y148V, Y148W, and H149M. The polymerases of the invention optionallycomprise one or more of these mutations. For example, in one aspect, thepolymerase is a Φ29-type polymerase that includes one or more mutationsin the N-terminal exonuclease domain (residues 5-189 as numbered withrespect to wild-type Φ29).

Making and Isolating Recombinant Polymerases

Generally, nucleic acids encoding a polymerase of the invention can bemade by cloning, recombination, in vitro synthesis, in vitroamplification and/or other available methods. A variety of recombinantmethods can be used for expressing an expression vector that encodes apolymerase of the invention. Methods for making recombinant nucleicacids, expression and isolation of expressed products are well known anddescribed in the art. A number of exemplary mutations and combinationsof mutations, as well as strategies for design of desirable mutations,are described herein. Methods for making and selecting mutations in theactive site of polymerases, including for modifying steric features inor near the active site to permit improved access by nucleotide analogsare found hereinabove and, e.g., in WO 2007/076057 POLYMERASES FORNUCLEOTIDE ANALOG INCORPORATION by Hanzel et al. and WO 2008/051530POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACID SEQUENCING byRank et al.

Additional useful references for mutation, recombinant and in vitronucleic acid manipulation methods (including cloning, expression, PCR,and the like) include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Kaufman et al. (2003) Handbook of Molecular andCellular Methods in Biology and Medicine Second Edition Ceske (ed) CRCPress (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley(ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al.(ed) PCR Cloning Protocols, Second Edition (Methods in MolecularBiology, volume 192) Humana Press; and in Viljoen et al. (2005)Molecular Diagnostic PCR Handbook Springer, ISBN 1402034032.

In addition, a plethora of kits are commercially available for thepurification of plasmids or other relevant nucleic acids from cells,(see, e.g., EasyPrep™, FlexiPrep™ both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Any isolatedand/or purified nucleic acid can be further manipulated to produce othernucleic acids, used to transfect cells, incorporated into relatedvectors to infect organisms for expression, and/or the like. Typicalcloning vectors contain transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular target nucleic acid.The vectors optionally comprise generic expression cassettes containingat least one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both.

Other useful references, e.g. for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Nucleic acids encoding the recombinant polymerases of the invention arealso a feature of the invention. A particular amino acid can be encodedby multiple codons, and certain translation systems (e.g., prokaryoticor eukaryotic cells) often exhibit codon bias, e.g., different organismsoften prefer one of the several synonymous codons that encode the sameamino acid. As such, nucleic acids of the invention are optionally“codon optimized,” meaning that the nucleic acids are synthesized toinclude codons that are preferred by the particular translation systembeing employed to express the polymerase. For example, when it isdesirable to express the polymerase in a bacterial cell (or even aparticular strain of bacteria), the nucleic acid can be synthesized toinclude codons most frequently found in the genome of that bacterialcell, for efficient expression of the polymerase. A similar strategy canbe employed when it is desirable to express the polymerase in aeukaryotic cell, e.g., the nucleic acid can include codons preferred bythat eukaryotic cell.

A variety of protein isolation and detection methods are known and canbe used to isolate polymerases, e.g., from recombinant cultures of cellsexpressing the recombinant polymerases of the invention. A variety ofprotein isolation and detection methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. New York (1990);Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollaget al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss, N.Y.; Walker(1996) The Protein Protocols Handbook Humana Press, N J, Harris andAngal (1990) Protein Purification Applications: A Practical Approach IRLPress at Oxford, Oxford, England; Harris and Angal Protein PurificationMethods: A Practical Approach IRL Press at Oxford, Oxford, England;Scopes (1993) Protein Purification: Principles and Practice 3^(rd)Edition Springer Verlag, N.Y.; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, N.Y.; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein. Additionaldetails regarding protein purification and detection methods can befound in Satinder Ahuj a ed., Handbook of Bioseparations, Academic Press(2000).

Kits

The present invention also features kits that incorporate thepolymerases of the invention, optionally with additional useful reagentssuch as one or more nucleotides and/or nucleotide analogs, e.g., forsequencing, nucleic acid amplification, or the like. Such kits caninclude the polymerase of the invention packaged in a fashion to enableuse of the polymerase (e.g., the polymerase immobilized in a ZMW array),optionally with a set of different nucleotide analogs of the invention,e.g., those that are analogous to A, T, G, and C, e.g., where one ormore of the analogs comprise a detectable moiety, to permitidentification in the presence of the analogs. Depending upon thedesired application, the kits of the invention optionally includeadditional reagents, such as natural nucleotides, a control template,and other reagents, such as buffer solutions and/or salt solutions,including, e.g., divalent metal ions such as Ca⁺⁺, Mg⁺⁺, Mn⁺⁺ and/orFe⁺⁺, and standard solutions, e.g., dye standards for detectorcalibration. Such kits also typically include instructions for use ofthe compounds and other reagents in accordance with the desiredapplication methods, e.g., nucleic acid sequencing, amplification andthe like.

Nucleic Acid and Polypeptide Sequences and Variants

As described herein, the invention also features polynucleotidesequences encoding, e.g., a polymerase as described herein. Examples ofpolymerase sequences that include features found herein, e.g., as inTables 3-6, are provided. However, one of skill in the art willimmediately appreciate that the invention is not limited to thespecifically exemplified sequences. For example, one of skill willappreciate that the invention also provides, e.g., many relatedsequences with the functions described herein, e.g., polynucleotides andpolypeptides encoding conservative variants of a polymerase of Tables3-6 or FIG. 7 or any other specifically listed polymerase herein.Combinations of any of the mutations noted herein or combinations of anyof the mutations herein in combination with those noted in otheravailable references relating to improved polymerases, such as Hanzel etal. WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION;Rank et al. WO 2008/051530 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCEDNUCLEIC ACID SEQUENCING; Hanzel et al. WO 2007/075987 ACTIVE SURFACECOUPLED POLYMERASES; Hanzel et al. WO 2007/075873 PROTEIN ENGINEERINGSTRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS; U.S.patent application publication 2010-0075332 ENGINEERING POLYMERASES ANDREACTION CONDITIONS FOR MODIFIED INCORPORATION PROPERTIES by PranavPatel et al.; U.S. patent application publication 2010-0093555 ENZYMESRESISTANT TO PHOTODAMAGE by Keith Bjornson et al.; U.S. patentapplication publication 2010-0112645 GENERATION OF MODIFIED POLYMERASESFOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCING by Sonya Clark etal.; U.S. patent application publication 2011-0189659 GENERATION OFMODIFIED POLYMERASES FOR IMPROVED ACCURACY 1N SINGLE MOLECULE SEQUENCINGby Sonya Clark et al.; and U.S. patent application publication2012-0034602 RECOMBINANT POLYMERASES FOR IMPROVED SINGLE MOLECULESEQUENCING are also features of the invention.

Accordingly, the invention provides a variety of polypeptides(polymerases) and polynucleotides (nucleic acids that encodepolymerases). Exemplary polynucleotides of the invention include, e.g.,any polynucleotide that encodes a polymerase of Tables 3-6 or FIG. 7 orotherwise described herein. Because of the degeneracy of the geneticcode, many polynucleotides equivalently encode a given polymerasesequence. Similarly, an artificial or recombinant nucleic acid thathybridizes to a polynucleotide indicated above under highly stringentconditions over substantially the entire length of the nucleic acid (andis other than a naturally occurring polynucleotide) is a polynucleotideof the invention. In one embodiment, a composition includes apolypeptide of the invention and an excipient (e.g., buffer, water,pharmaceutically acceptable excipient, etc.). The invention alsoprovides an antibody or antisera specifically immunoreactive with apolypeptide of the invention (e.g., that specifically recognizes afeature of the polymerase that confers decreased branching or increasedcomplex stability.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionally similarsequence are included in the invention. Variants of the nucleic acidpolynucleotide sequences, wherein the variants hybridize to at least onedisclosed sequence, are considered to be included in the invention.Unique subsequences of the sequences disclosed herein, as determined by,e.g., standard sequence comparison techniques, are also included in theinvention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid sequence. Similarly,“conservative amino acid substitutions,” where one or a limited numberof amino acids in an amino acid sequence (other than residues noted,e.g., in Tables 3-6 and FIG. 7 or elsewhere herein, as being relevant toa feature or property of interest) are substituted with different aminoacids with highly similar properties, are also readily identified asbeing highly similar to a disclosed construct. Such conservativevariations of each disclosed sequence are a feature of the presentinvention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid, while retaining the relevant mutational feature (forexample, the conservative substitution can be of a residue distal to theactive site region, or distal to an interdomain stability region). Thus,“conservative variations” of a listed polypeptide sequence of thepresent invention include substitutions of a small percentage, typicallyless than 5%, more typically less than 2% or 1%, of the amino acids ofthe polypeptide sequence, with an amino acid of the same conservativesubstitution group. Finally, the addition of sequences which do notalter the encoded activity of a nucleic acid molecule, such as theaddition of a non-functional or tagging sequence (introns in the nucleicacid, poly H is or similar sequences in the encoded polypeptide, etc.),is a conservative variation of the basic nucleic acid or polypeptide.

Conservative substitution tables providing functionally similar aminoacids are well known in the art, where one amino acid residue issubstituted for another amino acid residue having similar chemicalproperties (e.g., aromatic side chains or positively charged sidechains), and therefore does not substantially change the functionalproperties of the polypeptide molecule. The following sets forth examplegroups that contain natural amino acids of like chemical properties,where substitutions within a group is a “conservative substitution”.

TABLE 1 Conservative amino acid substitutions Positively NegativelyNonpolar and/or Polar, Charged Charged Aliphatic Side Uncharged AromaticSide Side Side Chains Side Chains Chains Chains Chains Glycine SerinePhenylalanine Lysine Aspartate Alanine Threonine Tyrosine ArginineGlutamate Valine Cysteine Tryptophan Histidine Leucine MethionineIsoleucine Asparagine Proline Glutamine

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention. In addition, target nucleic acids which hybridize to anucleic acid of the invention under high, ultra-high and ultra-ultrahigh stringency conditions, where the nucleic acids encode mutantscorresponding to those noted in Tables 3-6 and FIG. 7 or other listedpolymerases, are a feature of the invention. Examples of such nucleicacids include those with one or a few silent or conservative nucleicacid substitutions as compared to a given nucleic acid sequence encodinga polymerase of Tables 3-6 and FIG. 7 (or other exemplified polymerase),where any conservative substitutions are for residues other than thosenoted in Tables 3-6 and FIG. 7 or elsewhere as being relevant to afeature of interest (increased phototolerance, improved analog binding,etc.).

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least 50% as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least half as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Current Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2012); Hames andHiggins (1995) Gene Probes 1 IRL Press at Oxford University Press,Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) GeneProbes 2 IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determiningstringent hybridization and wash conditions, the hybridization and washconditions are gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents such as formalin in thehybridization or wash), until a selected set of criteria are met. Forexample, in highly stringent hybridization and wash conditions, thehybridization and wash conditions are gradually increased until a probebinds to a perfectly matched complementary target with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe probe to an unmatched target.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In some aspects, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid that encodes a polymerase of Tables3-6 and FIG. 7 or others described herein. The unique subsequence may beunique as compared to a nucleic acid corresponding to, e.g., a wild typeΦ29-type polymerase. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polymerase of Tables 3-6 and FIG. 7 or otherwisedetailed herein. Here, the unique subsequence is unique as compared to,e.g., a wild type Φ29-type polymerase or previously characterizedmutation thereof.

The invention also provides for target nucleic acids which hybridizeunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from the modifiedpolymerase sequences of the invention, wherein the unique subsequence isunique as compared to a polypeptide corresponding to a wild typeΦ29-type polymerase. Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or “percent identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a polymerase, or the aminoacid sequence of a polymerase) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90%, about95%, about 98%, about 99% or more nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Such “substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity over 50, 100, 150 or more residues is routinely usedto establish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% or moreidentity, can also be used to establish homology. Methods fordetermining sequence similarity percentages (e.g., BLASTP and BLASTNusing default parameters) are described herein and are generallyavailable.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyCurrent Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., supplemented through 2012).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

For reference, the amino acid sequence of a wild-type Φ29 polymerase ispresented in Table 2, along with the sequences of several otherwild-type Φ29-type polymerases.

TABLE 2 Amino acid sequence of exemplary wild-type Φ29-type polymerases.Φ29 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMA SEQ ID NO: 1WVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLV DDTFTIK M2YMSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVM SEQ ID NO: 2EIQADLYFHNLKFDGAFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSMGKPKPVQVNGGVVLVDS VFTIK B103MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVM SEQ ID NO: 3EIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVD SVFTIK GA-1MARSVYVCDFETTTDPEDCRLWAWGWMDIYNTDKWSYGEDIDSFMEWA SEQ ID NO: 4LNSNSDIYFHNLKFDGSFILPWWLRNGYVHTEEDRTNTPKEFTTTISGMGQWYAVDVCINTRGKNKNHVVFYDSLKKLPFKVEQIAKGFGLPVLKGDIDYKKYRPVGYVMDDNEIEYLKHDLLIVALALRSMFDNDFTSMTVGSDALNTYKEMLGVKQWEKYFPVLSLKVNSEIRKAYKGGFTWVNPKYQGETVYGGMVFDVNSMYPAMMKNKLLPYGEPVMFKGEYKKNVEYPLYIQQVRCFFELKKDKIPCIQIKGNARFGQNEYLSTSGDEYVDLYVTNVDWELIKKHYDIFEEEFIGGFMFKGFIGFFDEYIDRFMEIKNSPDSSAEQSLQAKLMLNSLYGKFATNPDITGKVPYLDENGVLKFRKGELKERDPVYTPMGCFITAYARENILSNAQKLYPRFIYADTDSIHVEGLGEVDAIKDVIDPKKLGYWDHEATFQRARYVRQKTYFIETTWKENDKGKLVVCEPQDATKVKPKIACAGMSDAIKERIRFNEFKIGYSTHGS LKPKNVLGGVVLMDYPFAIKAV-1 MVRQSTIASPARGGVRRSHKKVPSFCADFETTTDEDDCRVWSWGIIQVGK SEQ ID NO: 5LQNYVDGISLDGFMSHISERASHIYFHNLAFDGTFILDWLLKHGYRWTKENPGVKEFTSLISRMGKYYSITVVFETGFRVEFRDSFKKLPMSVSAIAKAFNLHDQKLEIDYEKPRPIGYIPTEQEKRYQRNDVAIVAQALEVQFAEKMTKLTAGSDSLATYKKMTGKLFIRRFPILSPEIDTEIRKAYRGGFTYADPRYAKKLNGKGSVYDVNSLYPSVMRTALLPYGEPIYSEGAPRTNRPLYIASITFTAKLKPNHIPCIQIKKNLSFNPTQYLEEVKEPTTVVATNIDIELWKKHYDFKIYSWNGTFEFRGSHGFFDTYVDHFMEIKKNSTGGLRQIAKLHLNSLYGKFATNPDITGKHPTLKDNRVSLVMNEPETRDPVYTPMGVFITAYARKKTISAAQDNYETFAYADTDSLHLIGPTTPPDSLWVDPVELGAWKHESSFTKSVYIRAKQYAEEIGGKLDVHIAGMPRNVAATLTLEDMLHGGTWNGKLIPVRVPGGTVLKDTTFTLKID CP-1MTCYYAGDFETTTNEEETEVWLSCFAKVIDYDKLDTFKVNTSLEDFLKSLY SEQ ID NO: 6LDLDKTYTETGEDEFIIFFHNLKFDGSFLLSFFLNNDIECTYFINDMGVWYSITLEFPDFTLTFRDSLKILNFSIATMAGLFKMPIAKGTTPLLKHKPEVIKPEWIDYIHVDVAILARGIFAMYYEENFTKYTSASEALTEFKRIFRKSKRKFRDFFPILDEKVDDFCRKHIVGAGRLPTLKHRGRTLNQLIDIYDINSMYPATMLQNALPIGIPKRYKGKPKEIKEDHYYIYHIKADFDLKRGYLPTIQIKKKLDALRIGVRTSDYVTTSKNEVIDLYLTNFDLDLFLKHYDATIMYVETLEFQTESDLFDDYITTYRYKKENAQSPAEKQKAKIMLNSLYGKFGAKIISVKKLAYLDDKGILRFKNDDEEEVQPVYAPVALFVTSIARHFIISNAQENYDNFLYADTDSLHLFHSDSLVLDIDPSEFGKWAHEGRAVKAKYLRSKLYIEELIQEDGTTHLDVKGAGMTPEIKEKITFENFVIGATFEGKRASKQIKGGTLIYETTFKIRETDYLVExemplary Mutation Combinations

A list of exemplary polymerase mutation combinations, and optionalcorresponding exogenous or heterologous features at the N- and/orC-terminal region of the polymerase, is provided in Tables 3 and 4.Positions of amino acid substitutions are identified relative to awild-type Φ29 DNA polymerase (SEQ ID NO:1) for the recombinantpolymerases in Table 3 and relative to a wild-type M2Y DNA polymerase(SEQ ID NO:2) for the recombinant polymerases in Table 4. Polymerases ofthe invention (including those provided in Tables 3 and 4) can includeany exogenous or heterologous feature (or combination of such features),e.g., at the N- and/or C-terminal region. For example, it will beunderstood that polymerase mutants in Tables 3 and 4 that do notinclude, e.g., a C-terminal polyhistidine tag can be modified to includea polyhistidine tag at the C-terminal region, alone or in combinationwith any of the exogenous or heterologous features described herein.Similarly, some or all of the exogenous features listed in Tables 3 and4 can be omitted, or substituted or combined with any of the otherexogenous features described herein, and still result in a polymerase ofthe invention. As will be appreciated, the numbering of amino acidresidues is with respect to a particular reference polymerase, such asthe wild-type sequence of the Φ29 polymerase (SEQ ID NO:1); actualposition of a mutation within a molecule of the invention may vary basedupon the nature of the various modifications that the enzyme includesrelative to the wild type Φ29 enzyme, e.g., deletions and/or additionsto the molecule, either at the termini or within the molecule itself.

TABLE 3 Exemplary mutations introduced into a Φ29 DNA polymerase.Positions are identified relative to SEQ ID NO: 1. N-terminal regionC-terminal region feature(s) Mutations feature(s) K131E Y148I Y224KE239G V250I L253A E375Y His10 A437G A484E D510K K512Y E515Q GGGSGGGSGGGSBtagV7 K135Q Y148I Y224K E239G V250I L253A E375Y His10 A437G A484E D510KK512Y E515Q GGGSGGGSGGGS BtagV7 K131E Y148I Y224K D235E E239G V250AL253H His10 E375Y A437G A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7Y148I Y224K E239G L253S E375Y A437G A484E His10 D510K K512Y E515QGGGSGGGSGGGS BtagV7 Y148I Q183F D235E E239G L253H E375Y A437G His10A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7 BtagV7 His10 Y148I Y224KE239G V250I L253H E375Y A437G His10 A484E D510K K512Y Y148I Y224K E239GV250I L253A E375Y A437G His10 A484E D510K K512Y E515Q GGGSGGGSGGGSBtagV7 K131E Y148I Y224K D235E E239G L253H E375Y His10 A437G A484E D510KK512Y E515Q GGGSGGGSGGGS BtagV7 Y148I Y224K D235E E239G L253H E375YA437G His10co BtagV7 A484E D510K K512Y E515Q Y148I Y224K E239G V250IL253A E375Y A437G His10 A484E D510K K512Y GGGSGGGSGGGS BtagV7 BtagV7His10 Y148I Y224K E239G V250I L253A E375Y A437G His10 A484E D510K K512YBtagV7 His10 K131E Y148I Y224K E239G V250I L253A E375Y His10 A484E D510KK512Y BtagV7 His10 K135Q Y148I Y224K E239G V250I L253A E375Y His10 A484ED510K K512Y BtagV7 His10 Y148I Y224K E239G L253H E375Y A437G A484E His10D510K K512Y K131E K135Q V141K L142K Y148I Y224K E239G His10 V250I L253AE375Y A437G A484E E508K D510K GGGSGGGSGGGS K512Y E515Q K536Q BtagV7K131E Y148I Y224K E239G V250I L253A E375Y His10 A437G A484E E508K D510KK512Y E515Q GGGSGGGSGGGS BtagV7 BtagV7 His10 K131Q Y148I Y224K E239GV250I L253A E375Y His10 A484E D510K K512Y

TABLE 4 Exemplary mutations introduced into an M2Y DNA polymerase.Positions are identified relative to SEQ ID NO: 2. N-terminal regionC-terminal region feature(s) Mutations feature(s) BtagV7 L250A S253AE372Y A481E K509Y His10 His10 Y145I E236G V247I L250A S253A His10 E372YA434G A481E D507K K509Y GGGSGGGSGGGS E512Q BtagV7 BtagV7 K132Q Y145IE236G V247I L250A His10 His10 E372Y A434G A481E D507K K509Y E512Q

The amino acid sequences of recombinant Φ29 and M2Y polymerasesharboring the exemplary mutation combinations of Tables 3 and 4 areprovided in Tables 5 and 6. Table 5 includes the polymerase portion ofthe molecule as well as the one or more exogenous features at the N-and/or C-terminal region of the polymerase, while Table 6 includes theamino acid sequence of the polymerase portion only.

TABLE 5Amino acid sequences of exemplary recombinant Φ29 and M2Y polymerasesincluding N- and C-terminal exogenous features. Amino acid positionsare identified relative to SEQ ID NO: 1 for recombinant Φ29 polymerases(denoted by “Phi29”) or relative to SEQ ID NO: 2 for recombinant M2Ypolymerases (denoted by “M2”). SEQ ID NO Amino Acid Sequence 7MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y148I_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK D510K_K512Y_E515Q.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK His10.GGGSGGGSGGGS.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 8MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K135Q_Y148I_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVLK D510K_K512Y_E515Q.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK His10.GGGSGGGSGGGS.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 9MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y148I_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI D235E_E239G V250A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253H_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_D510K_K512Y_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 10MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y148I_Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI L253S_E375Y_A437G_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A484E_D510K_K512Y_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK E515Q.His10.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK GGGSGGGSGGGS.BtagV7QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSSYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 11MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y148I_Q183F_D235E_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_L253H_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_D510K_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y_E515Q.His10.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK GGGSGGGSGGGS.BtagV7FGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 12MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.Phi29.Y148I_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY Y224K_E239G_V250I_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN L253H_E375Y_A437G_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ A484E_D510K_K512Y.WYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKD His10FKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDINSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH 13 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKK512Y_E515Q.His10. GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKGGGSGGGSGGGS.BtagV7 QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 14MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y148I_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI D235E_E239G_L253H_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK D510K_K512Y_E515Q.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK His10.GGGSGGGSGGGSQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD BtagV7KEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 15MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y148I_Y224K_D235E_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_L253H_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_D510K_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y_E515Q.His10.GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK BtagV7QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 16MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y148I_Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V250I_L253A_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_D510K_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y.His10.GGGSGGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK GGSGGGS.BtagV7QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 17MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.CTerm_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY His10.Phi29.Y148I_Y224K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN E239G_V250I_L253A_E375Y_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ A437G_A484E_D510K_WYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKD K512YFKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH 18 MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.CTerm_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY His10.Phi29.K131E_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN Y148I_Y224K_E239G_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ V250I_L253A_E375Y_WYMIDICLGYKGKRKIHTVIYDSLKKLPFPVEKIAKD A484E_D510K_K512YFKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRITYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH 19 MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.CTerm_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY His10.Phi29.K135Q_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN Y148I_Y224K_E239G_V250I_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ L253A_E375Y_A484E_D510K_WYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAQD K512YFKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH 20 MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.CTerm_GHIEGRHMSRKMFSCDFETTTKLDDCRVWAYGYME His10.M2.L250A_S253A_IGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDG E372Y_A481E_K509YAFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLS LPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASN PDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVDGYLKECSPDEATTTKFSVKCAGMTDTIKKKVTF DNFAVGFSSMGKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHH 21 MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.CTerm_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY His10.Phi29.Y148I_Y224K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN E239G_L253H_E375Y_A437G_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ A484E_D510K_K512YWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH 22 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNY M2.Y145I_E236G_V247I_KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL L250A_S253A_E372Y_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYK A434G_A481E_D507K_GKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDI K509Y_E512Q.His10.GGHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDR GSGGGSGGGS.BtagV7MTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLK DDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSM GKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 23 MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSSBtagV7.His10.M2.K132Q_ GHIEGRHMSRKMFSCDFETTTKLDDCRVWAYGYMEY145I_E236G_V247I_ IGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGL250A_E372Y_A434G_ AFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIA481E_D507K_K509Y_ DICFGYKGKRKLHTVIYDSLKKLPFPVKKIAQDFQLP E512Q.His10LLKGDIDIHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFI DKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFD NFAVGFSSMGKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHH 24 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_K135Q_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V141K_L142K_Y148I_Y224K_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E239G_V250I_L253A_LGYKGKRKIHTVIYDSLKKLPFPVEKIAQDFKLTKKK E375Y_A437G_A484E_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E508K_D510K_K512Y_E515Q_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD K536Q.His10.GGGKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP SGGGSGGGS.BtagV7AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKKVKGY LVQGSPDDYTDIKFSVKCAGMTDQIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 25MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y148I_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK E508K_D510K_K512Y_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.GGGSGGGQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD SGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKKVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 26MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSS BtagV7.His10.Phi29.K131Q_GHIEGRHMKHMPRKMYSCDFETTTKVEDCRVWAY Y148I_Y224K_E239G_GYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHN V250I_L253A_E375Y_LKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQ A484E_D510K_K512Y.WYMIDICLGYKGKRKIHTVIYDSLKKLPFPVQKIAKD His10FKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMV FDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRITYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHHHHH

TABLE 6Amino acid sequences of exemplary recombinant Φ29 and M2Y polymerases.Amino acid positions are identified relative to SEQ ID NO: 1 forrecombinant Φ29 polymerases (denoted by “Phi29”) or relative toSEQ ID NO: 2 for recombinant M2Y polymerases (denoted by “M2”).SEQ ID NO Amino Acid Sequence 27 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKD510K_K512Y_E515Q GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 28 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVLKD510K_K512Y_E515Q GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 29 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFID235E_E239G_V250A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253H_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_D510K_K512Y_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 30 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL253S_E375Y_A437G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA484E_D510K_K512Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK E515QGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSSYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 31 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Q183F_D235E_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_L253H_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y_E515QGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKFGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 32 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253H_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 33 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y_E515QGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 34 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFID235E_E239G_L253H_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKD510K_K512Y_E515Q GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 35 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_D235E_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_L253H_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512Y_E515QGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 36 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 37 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y148I_Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 38 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K13IE_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 39 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 40 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYM2.L250A_S253A_E372Y_ KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL A481E_K509YEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIR KAYRGGFTWLNDKYKEKEIGEGMVFDVNSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKV PYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVDGYLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGF SSMGKPKPVQVNGGVVLVDSVFTIK 41MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y148I_Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI L253H_E375Y_A437G_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A484E_D510K_K512YLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 42 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYM2.Y145I_E236G_V247I_ KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLL250A_S253A_E372Y_ EQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKA434G_A481E_D507K_ GKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDI K509Y_E512QHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLK DDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSM GKPKPVQVNGGVVLVDSVFTIK 43MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNY M2.K132Q_Y145I_E236G_KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL V247I_L250A_E372Y_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYK A434G_A481E_D507K_GKRKLHTVIYDSLKKLPFPVKKIAQDFQLPLLKGDIDI K509Y_E512QHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLK DDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSM GKPKPVQVNGGVVLVDSVFTIK 44MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_K135Q_V141K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI L142K_Y148I_Y224K_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E239G_V250I_L253A_LGYKGKRKIHTVIYDSLKKLPFPVEKIAQDFKLTKKK E375Y_A437G_A484E_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E508K_D510K_K512Y_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD E515Q_K536QKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKKVKGY LVQGSPDDYTDIKFSVKCAGMTDQIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 45 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKE508K_D510K_K512Y_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKKVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 46 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131Q_Y148I_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A484E_D510K_ LGYKGKRKIHTVIYDSLKKLPFPVQKIAKDFKLTVLK K512YGDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

Additional exemplary polymerase mutations and/or combinations thereofare provided in FIG. 7. In FIG. 7, positions of the mutations areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1)where the name of the polymerase includes “Phi29,” and where the name ofthe polymerase includes “M2” positions are identified relative to awild-type M2Y polymerase (SEQ ID NO:2). Where the feature “top( ) Vfusion” is listed, it indicates that the polymerase includes a fusion asdescribed in de Vega et al. (2010) “Improvement of φ29 DNA polymeraseamplification performance by fusion of DNA binding motifs” Proc NatlAcad Sci USA 107:16506-16511. Where the feature “Maltose Binding FusionProtein” is listed, it indicates that the polymerase includes a fusionwith maltose binding protein as known in the art. The notation“pET16.BtagV7co.His10co,” where the tags are listing in the N-terminalposition, indicates that the polymerase includes N-terminal biotin andHis10 tags. The feature “Cterm_His10co” is the same as listing the His10in the C terminal position; both terms indicate that the polymeraseincludes a C-terminal His10 tag. “pET16” or “pET11” refers to a vectorused to produce a recombinant Φ29 polymerase comprising the indicatedmutations, and “co” indicates that the polynucleotide sequence encodingcertain features (e.g., a His10 tag or BtagV7) has been codon optimized;neither notation is relevant to the structure of the polymerase.

The mutations or combinations of mutations shown in FIG. 7 are notlimited to use in a Φ29 or M2Y polymerase. Essentially any of thesemutations, any combination of these mutations, and/or any combination ofthese mutations with the other mutations disclosed or referenced hereincan be introduced into a polymerase (e.g., a Φ29-type polymerase) toproduce a modified recombinant polymerase in accordance with theinvention. Similarly, polymerases of the invention including themutations or mutation combinations provided in FIG. 7 can include anyexogenous or heterologous feature (or combination of such features),e.g., at the N- and/or C-terminal region. Similarly, some or all of theexogenous features listed in FIG. 7 can be omitted, or substituted orcombined with any of the other exogenous features described herein, andstill result in a polymerase of the invention. As will be appreciated,the numbering of amino acid residues is with respect to a particularreference polymerase, such as the wild-type sequence of the Φ29polymerase (SEQ ID NO:1) or M2Y polymerase (SEQ ID NO:2); actualposition of a mutation within a molecule of the invention may vary basedupon the nature of the various modifications that the enzyme includesrelative to the wild type Φ29 enzyme, e.g., deletions and/or additionsto the molecule, either at the termini or within the molecule itself.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Accordingly, the following examples areoffered to illustrate, but not to limit, the claimed invention.

Example 1 Characterization of Exemplary Recombinant Polymerases inSingle Molecule Sequencing Reactions

Recombinant polymerases based on Φ29 or M2Y polymerase and includingvarious combinations of mutations were expressed and purified asdescribed below. The polymerases were characterized by use in singlemolecule sequencing. Single molecule sequencing data was obtained withrecombinant Φ29 and M2Y polymerases including the mutation combinationslisted in FIG. 7. Exemplary data are presented in Table 7. Data for eachpolymerase is presented along with data for a control polymerase,acquired from the same chip for comparison. nReads represents the numberof ZMWs from which single molecule sequencing data was obtained.Accuracy and readlength are determined using data for those readsmeeting selected performance criteria.

TABLE 7 Single molecule sequencing with the exemplary recombinant Φ29and M2Y polymerases listed in Tables 3-5. Read Accuracy Control ControlControl Control Pol.^(a) nReads length^(b) (%) Pol.^(c) nReadsReadlength Accuracy 7 2696 1893 85.5 13 3089 1677 84.9 8 1958 1907 83.513 1927 1628 83.1 9 2324 2655 81.9 14 2623 2358 81.7 10 1782 1805 82 221434 1587 82.1 11 2278 3111 81.7 15 2481 2284 83.1 12 1347 1815 80.4 C12701 1207 83.8 13 2570 1744 85 17 2479 1823 83.4 14 1802 2089 83.5 154921 1915 83.1 15 2585 1617 83.6 C1 1886 1029 83.8 16 1264 2076 84.5 171400 1981 83.7 17 2123 1507 84.9 C1 1715 1145 85.1 18 2134 1289 84.3 C12282 1145 84.3 19 3001 1450 84.9 C1 2072 1261 84.8 20 868 976 82.8 C22231 908 83.8 21 2540 1470 81.2 C1 972 878 83.3 22 2119 2063 82.7 131802 2180 83.5 23 1772 996 82.8 C3 817 870 82.5 24 2330 2020 83  7 13762063 83.1 25 2644 1847 83  7 1427 1747 83.7 26 2098 1333 83.4 C1 20801197 83.4 ^(a)SEQ ID NO of exemplary polymerase (see Table 5).^(b)Readlength in nucleotides. ^(c)SEQ ID NO of control polymerase (seeTable 5). Additional control polymerases are C1: Φ29 BtagV7 His10 Y148IY224K E239G V250I L253A E375Y A484E D510K K512Y His10, C2: Φ29 BtagV7His10 L253A E375Y A484E K512Y His10, and C3: M2Y BtagV7 His10 Y145IE236G V247I L250A E372Y A434G A481E D507K K509Y E512Q His10, wherepositions are identified relative to SEQ ID NO: 1 for C1 and C2 andrelative to SEQ ID NO: 2 for C3.

Materials and Methods

Molecular Cloning

The phi29 and M2Y polymerase genes were cloned into either pET16 orpET11 (Novagen). Primers for specified mutations are designed andintroduced into the gene using the Phusion Hot Start DNA Polymerase Kit(New England Biolabs). A PCR reaction is performed to incorporatemutations and product is purified using ZR-96 DNA Clean andConcentration Kits (Zymo Research). PCR products are digested withNdeI/BamHI and ligated into the vector. Plasmids are transformed intoTOP10 E. coli competent cells, plated on selective media and incubatedat 37° C. overnight. Colonies are selected and plasmid is purified usingQiagen miniprep kits. Plasmids are then sequenced (Sequetech).

Protein Purification

Plasmid containing the recombinant polymerase gene is transformed intoBL21 Star21 CDE3+Biotin Ligase cells (Invitrogen) using heat shock.Transformed cells are grown in selective media overnight at 37° C. 200μL of the overnight culture are diluted into 4 mL of Overnight ExpressInstant TB Medium (EMD Chemicals) and grown at 37° C. until controlsreach O.D. value of 4-6. Cultures are then incubated at 18° C. for 16hours. Following this incubation, cells are harvested, resuspended inbuffer, and frozen at −80° C. Cells are thawed. The resulting lysate iscentrifuged and supernatant is collected. Polymerase is purified overnickel followed by heparin columns. The resulting proteins are run ongels and quantified by SYPRO® staining.

Single Molecule Sequencing

Enzymes are characterized by single molecule sequencing basically asdescribed in Eid et al. (2009) Science 323:133-138 (includingsupplemental information), using reagents similar to those commerciallyavailable in SMRT™ sequencing kits (Pacific Biosciences of California,Inc.). Each enzyme is initially screened with a single 5-7 minute movie,followed by secondary screening with 30 minute replicates whereapplicable. Data presented in Table 7 are from 30 minute movies. Enzymesare evaluated, e.g., based on readlength and accuracy compared tocontrol enzymes.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A composition comprising Φ29-type (phi29-type) recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position K131, an amino acid substitution at position K132, a K135Q substitution, a K135S substitution, an H149D substitution, a Q183F substitution, a G197D substitution, a G197E substitution, an 1201E substitution, a K206E substitution, an A437N substitution, and a D510S substitution, wherein identification of positions is relative to SEQ ID NO: 1, and wherein said polymerase exhibits polymerase activity.
 2. The composition of claim 1, wherein the recombinant polymerase comprises a K131E substitution or a K131Q substitution, wherein identification of positions is relative to SEQ ID NO:1.
 3. The composition of claim 1, wherein the recombinant polymerase comprises one or more mutation or combination of mutations selected from the group consisting of an amino acid substitution at position 253, an amino acid substitution at position 375, an amino acid substitution at position 484, an amino acid substitution at position 512, an amino acid substitution at position 510, an amino acid substitution at position 148, an amino acid substitution at position 224, an amino acid substitution at position 239, an amino acid substitution at position 250, an amino acid substitution at position 437, an amino acid substitution at position 235, an amino acid substitution at position 515, an amino acid substitution at position 141, an amino acid substitution at position 142, an amino acid substitution at position 504, an amino acid substitution at position 508, an amino acid substitution at position 513, an amino acid substitution at position 523, an amino acid substitution at position 536, an amino acid substitution at position 539, an amino acid substitution at position 205, an amino acid substitution at position 472, an amino acid substitution at position 437 and an amino acid substitution at position 253, an amino acid substitution at position 508 and an amino acid substitution at position 510, an A437G substitution and an L253H substitution, an A437G substitution and an L253C substitution, a V250A substitution and an L253H substitution, an A437G substitution, a D235E substitution, an E515Q substitution, an E515P substitution, an E515K substitution, a V250A substitution, a V250I substitution, a Y148I substitution, a Y224K substitution, an E239G substitution, a V141K substitution, an L142K substitution, an E508K substitution, an E508K substitution and a D510S substitution, a K536Q substitution, a K539Q substitution, a K205E substitution, a K205D substitution, a K205A substitution, a K472A substitution, an E375Y substitution, a K512Y substitution, an A484E substitution, an L253A substitution, an L253C substitution, an L253S substitution, an L253H substitution, and a D510K substitution, wherein identification of positions is relative to SEQ ID NO:1.
 4. The composition of claim 1, wherein the recombinant polymerase comprises E375Y, A484E, and K512Y substitutions, wherein identification of positions is relative to SEQ ID NO:1.
 5. The composition of claim 1, wherein the recombinant polymerase comprises one of the following combinations of mutations a) through q) selected from the group consisting of: a) K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; b) K135Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; c) K131E, Y148I, Y224K, D235E, E239G, V250A, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; d) Y148I, Y224K, E239G, L253S, E375Y, A437G, A484E, D510K, K512Y, and E515Q; e) Y148I, Q183F, D235E, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; f) Y148I, Y224K, E239G, V250I, L253H, E375Y, A437G, A484E, D510K, and K512Y; g) Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, K512Y, and E515Q; h) K131E, Y148I, Y224K, D235E, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; i) Y148I, Y224K, D235E, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; j) Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, and K512Y; k) Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, D510K, and K512Y; l) K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A484E, D510K, and K512Y; m) K135Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A484E, D510K, and K512Y; n) Y148I, Y224K, E239G, L253H, E375Y, A437G, A484E, D510K, and K512Y; o) K131E, K135Q, V141K, L142K, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, E508K, D510K, K512Y, E515Q, and K536Q; p) K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, E508K, D510K, K512Y, and E515Q; and q) K131Q, Y148I, Y224K, E239G, V250I, L253A, E375Y, A484E, D510K, and K512Y; wherein identification of positions is relative to SEQ ID NO:1.
 6. The composition of claim 5, wherein the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1.
 7. The composition of claim 5, wherein the C-terminal region of the recombinant polymerase comprises a His 10 tag.
 8. The composition of claim 1, where the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1.
 9. The composition of claim 1, wherein the recombinant polymerase comprises one or more exogenous features at the C-terminal and/or N-terminal region of the polymerase.
 10. The composition of claim 9, wherein the recombinant polymerase comprises a biotin ligase recognition sequence and a polyhistidine tag.
 11. The composition of claim 1, comprising a phosphate-labeled nucleotide analog.
 12. The composition of claim 11, wherein the nucleotide analog comprises a fluorophore.
 13. The composition of claim 1, comprising a phosphate-labeled nucleotide analog and a DNA template, wherein the recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template.
 14. The composition of claim 1, wherein the composition is present in a DNA sequencing system.
 15. The composition of claim 14, wherein the sequencing system comprises a zero-mode waveguide.
 16. The composition of claim 15, wherein the recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form.
 17. The composition of claim 1, wherein the recombinant polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:27-29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NOs:43-46.
 18. A method of sequencing a DNA template, the method comprising: a) providing a reaction mixture comprising: the DNA template, a replication initiating moiety that complexes with or is integral to the template, the recombinant polymerase of claim 1, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, and one or more nucleotides and/or nucleotide analogs; b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA; and c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA.
 19. The method of claim 18, wherein the subjecting and identifying steps are performed in a zero mode waveguide.
 20. A method of making a DNA, the method comprising: (a) providing a reaction mixture comprising: a template, a replication initiating moiety that complexes with or is integral to the template, the recombinant polymerase of claim 1, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and one or more nucleotides and/or nucleotide analogs; and (b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.
 21. The method of claim 20, wherein the mixture is reacted in a zero mode waveguide.
 22. The method of claim 20, the method comprising detecting incorporation of at least one of the nucleotides and/or nucleotide analogs. 